Alpha-1 antitrypsin produced from yeast for use in the treatment of viral infections - Patent Application 20070233633

Abstract

The present invention relates to a recombinant alpha 1-antithricin (AAT) protein or fragment thereof expressed in genetically modified yeast for use in the treatment of viral infections and / or pulmonary inflammation. In one embodiment, the viral infection is a viral respiratory infection, preferably a coronavirus infection such as SARSCoV infection, more preferably SARS-CoV-2. The present invention further relates to a method for producing a recombinant AAT protein or fragment(s) thereof from a genetically modified yeast. In an embodiment, the method comprises culturing a genetically modified yeast comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination, expressing the recombinant AAT in the cultured yeast, and isolating the recombinant AAT from the culture. In an embodiment, the recombinant AAT produced from the yeast is prepared in a pharmaceutical composition, such as a solution, preferably suitable for nebulization and subsequent inhalation by a subject.

Description

[Technical field]

[0001] The present invention relates to the field of recombinant protein production and the treatment of pathologies associated with viral infections and / or pulmonary inflammation with said recombinant proteins.

[0002] One aspect of the present invention relates to a recombinant alpha 1-antithricin (AAT) protein or fragment thereof expressed in genetically modified yeast for use in the treatment of viral respiratory infections. In an embodiment, the viral infection is a coronavirus infection, such as a SARSCoV infection, more preferably SARS-CoV-2.

[0003] A further aspect of the present invention relates to a pharmaceutical composition comprising the recombinant AAT protein or a fragment thereof and a pharma- ceutical acceptable carrier.In an embodiment, the pharmaceutical composition is suitable for inhalation by a subject.In an embodiment, the composition is a solution suitable for inhalation, a soluble powder suitable for inhalation, or a dry powder suitable for inhalation.

[0004] A further aspect of the invention relates to a method for producing a recombinant AAT protein or fragment(s) thereof from a genetically modified yeast. In an embodiment, the method comprises culturing a genetically modified yeast comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination, expressing the recombinant AAT in the cultured yeast, and isolating the recombinant AAT from the culture. In an embodiment, the recombinant AAT protein or fragment thereof is expressed in a yeast of the Saccharomycetaceae family, preferably Saccharomycetaceae Pichia, more preferably Pichia pastoris.

[0005] A further aspect of the invention relates to a method of making a pharmaceutical composition comprising producing recombinant AAT or fragment(s) thereof from a genetically modified yeast and adding one or more pharma- ceutical acceptable carriers to the recombinant AAT protein or fragment(s) thereof. [Background technology]

[0006] Severe Acute Respiratory Syndrome (SARS) is a coronavirus (CoV)-mediated respiratory disease that was first observed in 2002. Based on scientific reports, it is assumed that all human CoVs may be of zoonotic origin. Once infecting humans, the virus can spread rapidly by droplet transmission and close human-to-human contact, leading to epidemic scenarios and even pandemics. As an example, “coronavirus disease 2019” (COVID-19) is caused by the pathogenic coronavirus SARS-CoV-2. Since December 2019, the virus has spread worldwide in a matter of weeks, causing an international health emergency. The global pandemic poses major challenges to health systems, and even leads to restrictions on social life and weakening of the global market economy. As no proven effective treatment has been established to date and the disease is associated with high morbidity and mortality, there is a great need for therapeutic interventions for sick individuals, as well as preventive measures to contain the outbreak.

[0007] Alpha-1 antitrypsin (A1AT, AAT, PI, also known as SERPINA1) is a glycoprotein of approximately 52 kDa and one of the most abundant endogenous serine protease inhibitors (SERPIN superfamily). AAT is considered to be an acute phase protein, and AAT concentrations can increase many-fold during acute inflammation.

[0008] Although AAT is primarily known for its antiprotease and anti-inflammatory activities, studies carried out over the past decade have cumulatively demonstrated that AAT is also an immunomodulator and cytoprotective molecule. Thus, AAT-enriched microenvironments have been shown to include reduced levels of proinflammatory cytokines such as IL-1, IL-6, and TNF-α, and increased levels of anti-inflammatory mediators such as IL-1 receptor antagonist and IL-10. This phenomenon was shown not only in human PBMC in vitro studies, but also in samples obtained from cystic fibrosis patients who received inhaled AAT. At the same time, AAT has been demonstrated to directly bind to IL-8 and danger-associated molecular pattern molecules (DAMPs), such as extracellular HSP70 and gp96, which act as adjuvants of otherwise relevant immune responses (Non-Patent Document 1).

[0009] Surprisingly, the anti-inflammatory properties of AAT still allow innate immune cells to respond to real threats: macrophages readily phagocytose bacteria, neutrophils decontaminate the site of infection, and antigen-loaded dendritic cells migrate to draining lymph nodes. Meanwhile, T lymphocytes respond indirectly to the AAT-rich environment, waiting for stimuli from innate immune cells. For example, AAT has been shown to induce semi-mature antigen-presenting cells that favor the expansion of defensive regulatory T cells. B lymphocytes, belonging to both the innate and adaptive immune systems, appear to exert a modified response in the presence of AAT in the form of reduced isotype switching, resulting in enhanced protective IgM production. For example, as shown in CF patients during treatment with AAT, it has been suggested that the reduction in bacterial load is associated with enhanced anti-pathogen immune responses, and at the same time, local tissues may be spared from inappropriate excessive damage that could promote harmful adaptive responses by increasing the levels of local DAMPs (Non-Patent Document 1).

[0010] Reports have shown that AAT production is increased in COVID-19, but this anti-inflammatory response is overwhelmed in severe disease (Non-Patent Document 2). SARS-CoV-2 entry into infected cells is mediated by binding of the viral spike protein through its receptor-binding domain (RBD) to the human angiotensin-converting enzyme-2 (ACE2) target receptor. Blocking this interaction, for example by human antibodies, neutralizes the virus in patients and cures the infection. Further studies have also shown that SARS-CoV-2 entry is facilitated by endogenous and exogenous proteases. These proteases proteolytically activate the spike glycoprotein of SARS-CoV-2 and are key regulators of viral tropism. AAT has been identified as an abundant serum protease inhibitor that potently limits protease-mediated entry of SARS-CoV-2. AAT inhibition of protease-mediated SARS-CoV-2 entry in vitro occurs at concentrations below those present in serum and bronchoalveolar tissue, suggesting that the AAT effect is physiologically relevant (Non-Patent Document 3).

[0011] However, to date, obtaining AAT still relies heavily on purification from human plasma. For example, available methods are essentially limited to protein precipitation by addition of ammonium sulfate. A logical evolution in AAT purification has been the combination of ammonium sulfate fractionation with other procedures that can exploit the physicochemical properties of AAT. The first parameter considered by researchers was the charge of the protein to facilitate the separation of a large pool of proteins (typically the result of ammonium sulfate precipitation) into several smaller pools, one (or more) of which is enriched in AAT. More recently, with the advent of a wide range of advanced materials in the field of ion exchange and affinity chromatography, improved levels of purification have been observed. For example, Morihara et al. saturated human plasma with ammonium sulfate (80%), then dissolved the pellet in phosphate buffer at pH 8.0, dialyzed, and loaded onto an Affi-GEL Blue column. Two subsequent steps on a Zn-chelate column followed by DE-ion exchange chromatography produced homogeneous AAT.

[0012] Kwon et al. pioneered the purification of recombinant AAT from yeast, which was secreted into the medium as a glycosylated form. They developed a procedure that involved precipitation of the protein with ammonium sulfate (60%–75% saturation), followed by a series of subsequent chromatographic steps consisting of anion exchange chromatography (DEAE and monoQ columns) and affinity chromatography (A-Gel Blue column). Although yeast-produced AAT was fully functional as a protease inhibitor (compared to the plasma form), the molecular mass of this protein was reduced to that of recombinant AAT produced in Escherichia coli when treated with endoglycosidase H (unlike plasma AAT). This indicated that the N-linked glycosylation of this form was of the high mannose type. The authors also observed that glycosylation enhanced the kinetic stability of yeast-produced AAT against heat inactivation. However, Saccharomyces diastaticus is not ideal for secretory production of recombinant proteins (Non-Patent Document 4).

[0013] Non-Patent Document 5 discloses the expression and purification of recombinant human AAT in the methylotrophic yeast Pichia pastoris. Human AAT was secreted and expressed under the control of the inducible alcohol oxidase 1 (AOX1) promoter. The amount of AAT protein in the medium was measured to be 60 mg / L 72 hours after induction with methanol.

[0014] Non-Patent Document 6 discloses the use of P. pastoris as a host for efficient production and secretion of recombinant AAT. The findings revealed that optimizing the codon usage of the AAT gene for P. pastoris had a positive effect on the secretion level of AAT under the control of the inducible alcohol oxidase 1 (AOX1) and constitutive glycerol aldehyde phosphate dehydrogenase (GAP) promoters.

[0015] Patent Document 1 discloses a method for treating COVID-19 using a pharmaceutical composition containing recombinant human AAT in a dosage form suitable for inhalation. There are still many drawbacks to be addressed regarding the isolation of AAT from human plasma, such as the abundance of albumin in human plasma being a challenge and the limited yield of AAT from human plasma, requiring optimized methods for producing secreted recombinant protein. Furthermore, recombinant production of AAT in yeast has so far been limited by relatively low yields, resulting in a lack of clinical application.

[0016] Despite the progress in the purification of AAT from human plasma and the progress in the recombinant production of AAT, to the best of the inventors' knowledge, there has been no convincing solution to date to address the disadvantages of the prior art.There is a need for methods and means to reduce or avoid the problems associated with the isolation of AAT from human plasma, and there is a need for new means and uses of recombinant AAT. Research in this field is seeking new means to obtain the desired amount and purity level of AAT suitable for therapeutic use. Another objective problem that needs to be addressed is to provide a new method of treatment for pathologies associated with viral respiratory diseases and / or lung inflammation. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] International Publication No. 2021191900 [Non-patent literature]

[0018] [Non-Patent Document 1] Lior et al., Expert Opinion on TherapeuticPatents, 2016 [Non-Patent Document 2] McElvaney et al, Am J Respir Crit Care Med.2020 Sep 15; 202(6):812-821 [Non-Patent Document 3] Oguntuyo et al, bioRxiv. Aug 15; 2020 [Non-Patent Document 4] Purification and characterization of alpha1-antitrypsin secreted by recombinant yeast Saccharomyces diastaticus.J.Biotechnol. 1995, 42, 191-195. [Non-Patent Document 5] Arjmand et al. 2011 (Avicenna J MedBiotechnol.2011, 3(3):127-134) [Non-Patent Document 6] Arjmand et al. 2013 (Electronic Journal ofBiotechnology,2013, vol. 16, no. 1, 1-14) Summary of the Invention

[0019] In view of the prior art, the technical problem underlying the present invention is to provide an alternative and / or improved means for the treatment and / or prevention of pathologies associated with viral infections and / or lung inflammation. Another problem underlying the present invention is the provision of improved or alternative means for producing AAT in sufficient quantity and quality for therapeutic use. Another problem underlying the present invention was the provision of a novel means for treating coronavirus infections or pathologies associated with said infections.

[0020] These problems are solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0021] In one aspect, the present invention relates to recombinant alpha 1-antithricin (AAT) protein or a fragment thereof expressed in genetically modified yeast for use in the treatment of viral infection and / or pulmonary inflammation.

[0022] In one embodiment, the present invention relates to recombinant alpha 1-antithricin (AAT) protein or fragments thereof expressed in genetically modified yeast for use in the treatment of viral respiratory infections.

[0023] In an embodiment, the viral infection or viral respiratory infection is a coronavirus infection, such as a SARS CoV infection, more preferably SARS-CoV-2.

[0024] As demonstrated in the Examples below, the inventors have surprisingly found that recombinant AAT expressed in genetically modified yeast is highly suitable for the treatment of viral respiratory infections, in particular SARSCoV, such as SARS-CoV-2.

[0025] Although AAT production in yeast has been described previously, to the best of our knowledge, the efficacy of yeast-derived recombinant AAT in the treatment of viral respiratory diseases has not been previously evaluated.Surprisingly, the experimental studies presented in the following examples demonstrate that yeast-derived recombinant AAT can not only block virus entry into model cells in vitro, but also demonstrate improvements over existing therapeutic preparations of AAT purified from human plasma.For example, recombinant AAT isolated from yeast shows relatively superior properties in blocking the entry of viral pseudoparticles into Caco2 target cells compared to prolastin AAT preparations.

[0026] Thus, the present invention provides a novel and surprisingly effective recombinant AAT preparation using yeast as an expression system. Correspondingly, the isolated recombinant AAT protein itself, the genetically modified yeast used to produce recombinant AAT, and the methods of preparing and using the AAT produced in yeast, in embodiments, can be defined by the inventive findings of efficacy in treating viral respiratory diseases.

[0027] Embodiments relating to medical uses and medical indications: In one aspect, the present invention relates to recombinant alpha 1-antithricin (AAT) protein or fragments thereof expressed in genetically modified yeast for use in the treatment of viral respiratory infections.

[0028] In an embodiment, the present invention relates to recombinant alpha 1-antithricin (AAT) protein or fragments thereof expressed in genetically modified yeast for use in the treatment of pathologies associated with viral respiratory infections.

[0029] In one embodiment, the viral respiratory infection is a coronavirus infection, preferably a SARS coronavirus infection, more preferably SARS-CoV-2.

[0030] In an embodiment, the present invention relates to a recombinant alpha 1-antithricin (AAT) protein or a fragment thereof expressed in a genetically modified Pichia pastoris yeast strain for use in the treatment of viral respiratory infections.

[0031] Although AAT is known to be effective in treating pulmonary diseases, the use of AAT expressed in yeast is an unexpectedly useful and effective means for treating viral infections of the lungs or respiratory system.First, pulmonary diseases themselves are very different in terms of pathological causes, and viral pulmonary diseases are mechanistically and biologically different from other pulmonary diseases.The effectiveness of treating pulmonary viral diseases as in the present invention could not be expected from previous disclosures on AAT treatment for other pulmonary diseases, such as those based on non-viral inflammatory or chronic conditions, such as immune disorders or chronic obstructive pulmonary disease (COPD).

[0032] In the context of the present invention, the use of recombinant alpha 1-antithricin (AAT) protein or fragments thereof expressed in genetically modified yeast is not limited to the treatment of SARS-CoV infections, such as SARS-CoV-2. The viral infections to be treated may also refer in embodiments to other human coronavirus infections, such as human coronaviruses NL63, 229E, HKU1 and / or OC43, or coronaviruses derived therefrom.

[0033] In an embodiment, the condition being treated is a form of COVID-19, which may be asymptomatic, mild, moderate or severe.

[0034] In an embodiment, the recombinant AAT described herein is for use in the prevention and / or treatment of Long COVID or a pathology associated with Long COVID.

[0035] In an embodiment, the patient is additionally provided with standard treatments for COVID-19, including, for example, oxygenation, non-invasive ventilation, high-flow oxygen, mechanical ventilators, and extracorporeal membrane oxygenation.

[0036] In an embodiment, the patient is additionally provided with standard treatment for COVID-19 and / or other viral infections such as influenza and RSV, for example, one or more analgesics, antipyretics and / or anti-inflammatory agents may be administered in combination with the AAT treatment.

[0037] In embodiments, the patient being treated has COVID-19 symptoms that may be present for any duration or intensity, for example, the patient may present with acute, delayed and / or ongoing COVID symptoms.

[0038] In an embodiment of the invention, recombinant AAT is administered over several consecutive days, hi embodiments, a patient may receive inhalation therapy multiple times within a day, multiple times within a week, or even continuously over a period of several weeks.

[0039] In an embodiment of the invention, recombinant AAT is administered as a prophylactic treatment, for example to achieve short-term prevention of viral infection in the respiratory tract by administration of AAT immediately prior to exposure or potential exposure to the virus.

[0040] In embodiments of the invention, treatment results in at least one of the following outcomes: enhanced viral clearance, reduced hospitalization, reduced oxygen dependency, reduced need for intensive care or mechanical ventilation, reduced healthcare utilization or burden, reduced school or work absenteeism, reduced need for antibiotics, reduced need for steroids, reduced relapse frequency, and / or reduced morbidity or risk of morbidity.

[0041] In an embodiment, the duration that a subject exhibits symptoms of a viral respiratory disease, such as influenza or COVID, is shortened using the treatment of the present invention. The symptoms of the condition may be significantly reduced compared to when AAT is not administered. Thus, symptoms may be reduced to less than 2 weeks, less than 10 days, less than 7 days, or less than 6, 5, 4, 3, 2, or 1 day of viral respiratory disease symptoms.

[0042] In an embodiment, the AAT treatment additionally includes at least one other therapeutic agent. Such an agent may be, but is not limited to, an antiviral agent, such as an antiviral agent selected from the group consisting of a protease inhibitor, a helicase inhibitor, a viral replication inhibitor, and a viral cell entry inhibitor.

[0043] In an embodiment, the patient is additionally provided with an antiviral treatment selected from, but not limited to, a neuraminidase inhibitor (NAI) such as Tamiflu (oseltamivir), alpivab (peramivir), Relenza (zanamivir) or zanamivir (intravenous formulation), or one or more M2 inhibitors (adamantanes) such as amantadine or rimantadine.

[0044] In an embodiment, the present invention relates to recombinant alpha 1-antithricin (AAT) protein or a fragment thereof expressed in genetically modified yeast for use in the treatment or prevention of any given viral infection of the respiratory tract.

[0045] By way of example, respiratory viral diseases may result from any infection with adenovirus, coronavirus (common cold virus), influenza (flu) virus, parainfluenza virus, parvovirus B19 (fifth disease), respiratory syncytial virus (RSV), or rhinovirus (common cold). Additional viral respiratory diseases or viruses may be associated with HBoV, human bocavirus; HCoV, human coronavirus; HMPV, human metapneumovirus; HPIV, human parainfluenza virus; HRSV, human respiratory syncytial virus; HRV, human rhinovirus; PCF, pharyngoconjunctival fever; SARS, severe acute respiratory syndrome; SARS-CoV, SARS-associated coronavirus; URI, upper respiratory tract infection.

[0046] In an embodiment, the present invention also relates to a recombinant alpha 1-antithricin (AAT) protein or a fragment thereof expressed in genetically modified yeast for use in the treatment or prevention of influenza infection.

[0047] In an embodiment, the present invention also relates to recombinant alpha 1-antithricin (AAT) protein or fragments thereof expressed in genetically modified yeast for use in the treatment or prevention of viral respiratory disease, preferably those that are sensitive to protease inhibition as achieved by AAT, as shown in the examples below.

[0048] In one embodiment, recombinant AAT protein is administered to treat a viral infection such as SARS-CoV-2, influenza or RSV, the AAT protein or a fragment thereof is expressed from Pichia pastoris, and the AAT protein or a fragment thereof is administered by inhalation of a nebulized solution.

[0049] In one embodiment, recombinant AAT protein is administered to treat the viral infection SARS-CoV-2, and the AAT protein or a fragment thereof is expressed from Pichia pastoris and isolated using chromatography, such as ion exchange or hydrophobic interaction chromatography, and the AAT protein or a fragment thereof is administered by inhalation of a nebulized solution.

[0050] Embodiments relating to the AAT sequence: In one embodiment, the AAT protein or fragment thereof comprises a sequence according to SEQ ID NO:3 or SEQ ID NO:5, or is encoded by SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:4.

[0051] In the context of the present invention, SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5 are exemplary nucleotide sequences encoding AAT protein. The nucleotide sequence encoding AAT according to the present invention is not limited to SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5 and may in embodiments be optimized for expression, e.g., a codon-optimized nucleotide sequence encoding AAT protein.

[0052] In an embodiment, the AAT protein or a fragment thereof comprises or consists of a sequence according to SEQ ID NO: 3 or SEQ ID NO: 5. These sequences are exemplary sequences and are non-limiting for the present invention.

[0053] Sequence variations, including amino acid sequence changes, for example by substitutions and / or deletions, and / or length changes, are disclosed in more detail below.

[0054] Embodiments relating to AAT produced in yeast: As described in more detail herein, the recombinant AAT of the present invention is expressed from yeast.Although the therapeutic utility of isolated human AAT (derived from plasma) is known to those skilled in the art, the activity of yeast AAT protein in the specific context of treating viral respiratory diseases was not expected.The yeast origin of recombinant AAT as described herein not only allows the recombinant expression system in yeast to produce higher amounts of AAT, but also yeast-expressed AAT achieves unexpected therapeutic effects in the context of treating viral infections.As shown in the following examples, recombinant AAT from yeast appears to be more effective in blocking viral entry into target cells in vitro compared to AAT isolated from plasma.

[0055] Without being limited to the preferred and non-limiting examples, the yeast strains and expression means described herein may play a role in obtaining the beneficial properties of the recombinant yeast described herein in embodiments. The source of recombinant AAT may provide physical and / or functional properties that are unique to the therapeutic recombinant AAT protein itself, in terms of the yeast strain, expression vector, use of a particular promoter, or culture conditions. Thus, the characteristics related to the expression or production of recombinant AAT from yeast may be used in embodiments to define the recombinant AAT protein of the present invention.

[0056] In one embodiment, the recombinant AAT protein or fragment thereof is expressed in a yeast of the Saccharomycete family, preferably Saccharomycetaceae Pichia, more preferably Pichia pastoris.

[0057] In one embodiment, the recombinant AAT or fragment thereof has a serum half-life and / or activity equal to or greater than that of AAT purified from human plasma.

[0058] In one embodiment, the AAT or fragment thereof comprises a post-translational modification of a recombinant AAT protein or fragment thereof.

[0059] In one embodiment, the post-translational modification is O-glycosylation, N-glycosylation, N-terminal methionine removal, N-acetylation and / or phosphorylation, or any combination thereof, and preferably includes one or more specific modifications from expression in yeast, such as Man3GlcNAc2.

[0060] In one embodiment, the recombinant AAT protein or fragment thereof comprises one or more human-like glycoform patterns.

[0061] In one embodiment, the AAT protein or fragment thereof is a. culturing a genetically modified yeast comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination; b. expressing the recombinant AAT in a yeast culture; c. isolating the recombinant AAT from the culture; The method comprises producing the polypeptide from a genetically modified yeast.

[0062] In one embodiment, the AAT protein or fragment thereof is a. culturing a genetically modified yeast comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination, said yeast being preferably a yeast of the Saccharomycetaceae family, preferably Saccharomycetaceae Pichia, more preferably Pichia pastoris; b. expressing recombinant AAT or a fragment thereof in cultured yeast, the promoter being preferably glyceraldehyde-3-phosphate dehydrogenase (GAP) or alcohol oxidase I (AOX1); c. isolating the recombinant AAT or a fragment thereof from the culture; The method comprises producing the polypeptide from a genetically modified yeast.

[0063] Embodiments relating to methods for producing AAT from yeast: In another aspect, the present invention relates to a method for producing a recombinant alpha 1-antithricin (AAT) protein or fragment(s) thereof from a genetically modified yeast, comprising: a. culturing a genetically modified yeast comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination; b. expressing the recombinant AAT in a yeast culture; c. isolating the recombinant AAT from the culture; Includes.

[0064] The recombinant AAT obtained by the method for producing AAT as described herein is preferably adapted for medical use to treat viral infections, preferably viral respiratory infections.

[0065] In embodiments, the yeast used herein may be any yeast species suitable for expressing recombinant proteins.

[0066] In embodiments, the yeast is one or more of a Saccharomyces species, a Pichia species, a Hansenula species, a Yarrowia species, an Arxula species, a Kluyveromyces species, or a Schizosaccharomyces species, such as Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Arxula adeninivorans, Kluyveromyces lactis, or Schizosaccharomyces spp. lactis or Schizosaccharomyces pombe.

[0067] In an embodiment, the yeast is of the Saccharomycete family, preferably Saccharomycetaceae Pichia.

[0068] In an embodiment, the yeast is Pichia pastoris.

[0069] In an embodiment, the yeast is Saccharomyces cerevisiae.

[0070] In an embodiment, the yeast is Schizosaccharomyces pombe.

[0071] Without wishing to be bound by theory, the use of a yeast host to express recombinant AAT (preferably rhAAT) confers a structure, function and / or activity to the AAT that differs from AAT isolated from a human source, such as human plasma. Thus, the definition of AAT recombinantly expressed from yeast refers to the structural and / or functional characteristics of the AAT protein itself.

[0072] In embodiments, post-translational modifications of the AAT protein induced via expression in yeast allow for the combination of superior AAT activity and unusually high yields via expression and secretion into the culture supernatant.

[0073] In embodiments, expression in yeast allows for one or more of increased stability, increased purity, increased activity and / or increased resistance to degradation following administration of yeast AAT relative to concentrated and / or isolated human AAT preparations.

[0074] In embodiments, the AAT produced in yeast surprisingly shows little undesirable side effects after administration in human subjects.The inventors envision that the AAT expressed from yeast shows a good tolerability profile in human subjects and has a low risk of undesirable immune reaction to AAT preparations.

[0075] In embodiments, the particular yeast strain employed for recombinant AAT production confers a structure, function, and / or activity to AAT that differs from AAT produced from other yeast strains and / or AAT isolated from human sources such as human plasma. For example, the examples provided herein show unexpectedly successful expression, secretion, and isolation of recombinant AAT from Pichia pastoris. In embodiments, AAT expressed from Pichia pastoris exhibits one or more advantages in terms of structure, function, activity, expression, secretion, and / or isolation over expression of AAT in other yeast hosts and / or isolated human AAT.

[0076] In one embodiment, the methods used herein include the use of an inducible promoter configured to regulate expression of AAT in yeast, such as alcohol oxidase I (AOX1).

[0077] In an embodiment, the AAT protein or fragment(s) thereof is of an origin selected from the group consisting of mammalian, fish, fungal and plant, preferably the AAT protein is a human AAT protein or fragment thereof.

[0078] In embodiments, the AAT protein or fragment(s) are secreted from the yeast into the medium to a concentration of at least 1 mg protein per liter of liquid medium (mg / L), preferably to a concentration of 2 mg / L to 100 mg / L, more preferably to a concentration of 5 mg / L to 100 mg / L, or 10 mg / L to 100 mg / L.

[0079] In embodiments, the AAT protein or fragment(s) secreted from the yeast into the medium is at a concentration of 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, 55 mg / L, 60 mg / L, 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L, 100 mg / L. The yield can also be a range derived from any one or more of the values ​​provided above. In embodiments, the AAT protein or fragment(s) are secreted from the yeast into the medium at concentrations that exceed those presented herein.

[0080] In one embodiment, the genetically modified yeast is of the Saccharomycete family, preferably Saccharomycetaceae Saccharomyces, more preferably Saccharomyces cerevisiae, and the yeast also produces xanthohumol. In an embodiment, the genetically modified yeast is a Pichia strain, and also produces xanthohumol.

[0081] The genetically modified yeast used to express recombinant AAT preferably contains a yeast expression vector that is configured for efficient AAT expression. Suitable vectors that contain replication and expression components for yeast systems are known to those skilled in the art. Suitable vectors include, but are not limited to, those described herein, such as pGAPz, pPICz and / or pEX-A258.

[0082] As described herein, an exogenous nucleic acid molecule, such as a yeast expression vector, having an AAT coding region is employed, where the AAT coding region is operably linked to a promoter or promoter / enhancer combination. Suitable promoters can be selected by those skilled in the art.

[0083] In an embodiment, the promoter is glyceraldehyde-3-phosphate dehydrogenase (GAP).

[0084] In another embodiment, the promoter is inducible, preferably alcohol oxidase I (AOX1).

[0085] In an embodiment, expression of AAT from genetically modified yeast typically results in secretion of soluble AAT in the supernatant of yeast culture.Recombinant AAT typically requires isolation or purification before further use.Clinical grade AAT can also be produced using the required protocols.

[0086] In an embodiment, recombinant AAT of the present invention is isolated using ion exchange and / or affinity and / or hydrophobic interaction chromatography and / or size exclusion chromatography.In an embodiment, yeast culture supernatant may be treated with ammonium sulfate to precipitate protein.The precipitated pellet is then dissolved in a suitable buffer, such as a phosphate buffer at pH 8.0, and optionally dialyzed and loaded onto a suitable column for purification.For example, ion exchange chromatography can produce recombinant AAT with high purity.

[0087] In an embodiment, purification of the supernatant of recombinant AAT produced in yeast comprises cation exchange chromatography (CEX).

[0088] In an embodiment, purification of the supernatant of recombinant AAT produced in yeast comprises anion exchange chromatography (AEX).

[0089] In an embodiment, purification of the supernatant of recombinant AAT produced in yeast comprises size exclusion chromatography.

[0090] In an embodiment, purification of the supernatant of recombinant AAT produced in yeast comprises hydrophobic interaction chromatography.

[0091] In an embodiment, purification of the supernatant of recombinant AAT produced in yeast comprises affinity chromatography.

[0092] In an embodiment, AAT may be expressed in yeast as a fusion protein with a suitable purification tag, such as a histidine tag (His tag), which facilitates the subsequent purification step.

[0093] In embodiments, the method of preparing AAT from yeast can include modifying recombinant AAT protein in vitro after isolation from yeast. In embodiments, the modification includes covalently linking the recombinant AAT protein to a biocompatible polymer.

[0094] Composition and dosage form embodiments: A further aspect of the present invention relates to a pharmaceutical composition comprising the recombinant AAT protein or a fragment thereof produced in yeast and a pharma- ceutically acceptable carrier.

[0095] In an embodiment, the pharmaceutical composition is suitable for inhalation by a subject, hi an embodiment, the composition is a solution suitable for inhalation, a soluble powder suitable for inhalation, or a dry powder suitable for inhalation.

[0096] In an embodiment, administration by inhalation is via a single dose of recombinant AAT or a fragment thereof produced in yeast, via a solution suitable for inhalation, a soluble powder suitable for inhalation, or a dry powder suitable for inhalation. The above suctions may be included.

[0097] In one embodiment, the AAT protein or fragment thereof is administered by inhalation of a nebulized solution.

[0098] In embodiments, the pharmaceutical composition may be prepared by a nebulizer for inhalation, such as a Pari Boy Pro, or similar device.

[0099] In one embodiment, the viral infection is a coronavirus infection, such as SARS CoV, preferably SARS-CoV-2, and the AAT protein or fragment thereof is administered by inhalation of a nebulized solution.

[0100] In a related aspect, the invention relates to a pharmaceutical composition comprising recombinant alpha 1-antithricin (AAT) protein or a fragment thereof expressed in genetically modified yeast for use in the treatment of viral respiratory infections.

[0101] In one embodiment, the viral infection is SARS-CoV-2, the AAT protein or a fragment thereof is expressed in Pichia pastoris, and the AAT is administered by inhalation of a nebulized solution.

[0102] In embodiments, the compositions of the invention are formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0103] In embodiments, the pharmaceutical composition for use may involve the simultaneous or sequential administration of one or more other active agents useful in treating conditions associated with viral infection or pulmonary inflammation.

[0104] In one embodiment, the one or more other active agents may be xanthohumol.

[0105] Without being bound by theory, during the fermentation process with yeasts of the Saccharomycete family, preferably Saccharomycetaceae Saccharomyces, more preferably Saccharomyces cerevisiae, when hops are added to the process, xanthohumol is produced as a by-product and acts as a potent pan-inhibitor against various coronaviruses by targeting the proteases of coronaviruses, e.g., SARS-CoV-2.

[0106] Wang et al., 2021 show that pretreatment with xanthohumol restricted the replication of SARS-CoV-2 and PEDV in Vero-E6 cells, whereas xanthohumol inhibited protease activity in enzyme assays. Xanthohumol is a potent pan-inhibitor of coronaviruses and an excellent lead compound for further drug development (Wang et al., 2021, Xanthohumol Is a Potent Pan-Inhibitor ofCoronaviruses Targeting Main Protease, international journal of molecular sciences).

[0107] Thus, in an embodiment, a genetically modified yeast from the Saccharomycete family, preferably Saccharomycetaceae Saccharomyces, more preferably Saccharomyces cerevisiae, not only produces recombinant AAT protein but also produces the auxiliary substance xanthohumol, which may provide a synergistic effect for treating viral infections, such as coronavirus.

[0108] In an embodiment, the present invention relates to a method for preventing, reducing the risk of and / or treating a viral respiratory infection, comprising administering to a subject in need thereof a therapeutic amount of a recombinant AAT protein, wherein the recombinant AAT protein or a fragment thereof is expressed in a genetically modified yeast as described herein, and wherein the administration preferably comprises inhalation of the AAT protein, e.g., in aerosolized droplets, and / or injection of the AAT protein into the subject.

[0109] Further aspects of the present invention: Another aspect of the invention relates to a recombinant AAT protein or a fragment thereof obtainable from the method as described in any embodiment of the invention.

[0110] A further aspect of the present invention relates to a genetically modified yeast, the yeast being genetically modified to express recombinant AAT and fragment(s) thereof, comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination.

[0111] A further aspect of the invention relates to recombinant AAT protein or fragments thereof produced by expression in yeast.

[0112] A further aspect of the present invention is a. culturing a genetically modified yeast comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination, such as glyceraldehyde-3-phosphate dehydrogenase (GAP), alcohol oxidase I (AOX1), etc.; b. expressing recombinant AAT in yeast culture; c. isolating recombinant AAT from the culture; and The present invention relates to a recombinant AAT protein or fragment thereof produced by a process comprising:

[0113] A further aspect of the present invention is a. Producing recombinant AAT or fragment(s) thereof from a genetically modified yeast according to the methods described herein; b. adding one or more pharma- ceutical acceptable carriers to the recombinant AAT protein or fragment(s) thereof; The present invention relates to a method for producing a pharmaceutical composition comprising:

[0114] Further exemplary aspects and embodiments of the present invention relate, for example, to:

[0115] Embodiment 1. A method for producing recombinant alpha 1-antithricin (AAT) protein or fragment(s) thereof from a genetically modified yeast, comprising: a. culturing a genetically modified yeast comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination; b. expressing the recombinant AAT in the yeast culture; c. isolating recombinant AAT from the culture; A method comprising:

[0116] Embodiment 2. The method of the above embodiment, wherein the promoter is an inducible promoter configured to regulate expression of AAT in yeast, such as alcohol oxidase I (AOX1).

[0117] Embodiment 3. The method according to any one of the above embodiments, wherein said yeast is a yeast of the Saccharomycetaceae family, preferably Saccharomycetaceae Pichia, more preferably Pichia pastoris.

[0118] Embodiment 4. The method of any one of the above embodiments, wherein the AAT protein or fragment(s) thereof is of an origin selected from the group consisting of mammalian, fish, fungal and plant, and preferably, the AAT protein is a human AAT protein.

[0119] Embodiment 5. The method of any one of the above embodiments, wherein the exogenous nucleic acid molecule comprises the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

[0120] Embodiment 6. The method of any one of the preceding embodiments, wherein the recombinant AAT has a serum half-life and / or activity equal to or greater than AAT purified from human plasma.

[0121] Embodiment 7. The method of any one of the above embodiments, wherein the AAT protein or fragment(s) thereof is secreted from the yeast into the medium to a concentration of at least 1 mg protein per liter of liquid medium (mg / L), preferably to a concentration of 5 mg / L to 100 mg / L.

[0122] Embodiment 8 The method of any one of the preceding embodiments, comprising post-translational modification of the recombinant AAT protein.

[0123] Embodiment 9. The method of any one of the above embodiments, wherein the post-translational modification is O-glycosylation, N-glycosylation, N-terminal methionine removal, N-acetylation and / or phosphorylation, or any combination thereof.

[0124] Embodiment 10 The method of any one of the above embodiments, wherein the recombinant AAT protein has one or more human-like glycoform patterns.

[0125] Embodiment 11 The method of any one of the preceding embodiments, comprising modifying the recombinant AAT protein in vitro after isolation from the yeast.

[0126] Embodiment 12. The method according to the above embodiment, wherein the modification is covalent attachment of the recombinant AAT protein to a biocompatible polymer.

[0127] Embodiment 13. A recombinant AAT protein or a fragment thereof obtainable by the method according to any one of the above embodiments.

[0128] Embodiment 14. A recombinant AAT protein or fragment thereof according to the above embodiments, comprising one or more specific modifications from expression in yeast, such as Man3GlcNAc2.

[0129] Embodiment 15. A genetically modified yeast, the yeast being genetically modified to express recombinant AAT and fragment(s) thereof, comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination.

[0130] Embodiment 16 The genetically modified yeast of any of the preceding embodiments, wherein the exogenous nucleic acid molecule comprises the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

[0131] Embodiment 17. The genetically modified yeast of any one of embodiments 15 and 16, wherein the promoter is glyceraldehyde-3-phosphate dehydrogenase (GAP).

[0132] Embodiment 18. The genetically modified yeast according to any one of embodiments 15 to 17, wherein the promoter is inducible, preferably alcohol oxidase I (AOX1).

[0133] Embodiment 19. A genetically modified yeast according to any one of embodiments 15 to 18, wherein the yeast is a yeast of the family Saccharomycetaceae, preferably Saccharomycetaceae Pichia, more preferably Pichia pastoris.

[0134] EMBODIMENT 20. a. culturing a genetically modified yeast comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination, such as glyceraldehyde-3-phosphate dehydrogenase (GAP), alcohol oxidase I (AOX1), etc.; b. expressing recombinant AAT in yeast culture; c. isolating recombinant AAT from the culture; and 2. A recombinant AAT protein or a fragment thereof produced by a process comprising the steps of:

[0135] Embodiment 21. The recombinant AAT protein or fragment thereof according to the above embodiment, wherein the exogenous nucleic acid molecule comprises the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, and / or the AAT amino acid sequence comprises the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5.

[0136] Embodiment 22. A recombinant AAT protein or fragment thereof according to embodiments 20 to 21, comprising post-translational modifications, such as O-glycosylation, N-glycosylation, N-terminal methionine removal, N-acetylation and / or phosphorylation, and any combination thereof.

[0137] Embodiment 23. A recombinant AAT protein or fragment thereof according to any one of embodiments 20 to 22, comprising one or more human-like glycoform patterns.

[0138] EMBODIMENT 24. a. Producing recombinant AAT or a fragment thereof from a genetically modified yeast according to any one of embodiments 1 to 12; b. adding one or more pharma- ceutical acceptable carriers to the recombinant AAT protein or fragment(s) thereof; A method for producing a pharmaceutical composition comprising:

[0139] Embodiment 25. A pharmaceutical composition comprising a recombinant AAT protein or a fragment thereof and a pharma- ceutically acceptable carrier, wherein the recombinant AAT protein or a fragment thereof is expressed in a genetically modified yeast according to any one of embodiments 15 to 19.

[0140] Embodiment 26. A pharmaceutical composition according to any of the above embodiments, wherein the composition is a solution suitable for inhalation or a soluble powder suitable for inhalation or a dry powder for direct inhalation.

[0141] Embodiment 27. A pharmaceutical composition according to any of the above embodiments, wherein the composition can be prepared by a nebulizer for inhalation.

[0142] Embodiment 28. A pharmaceutical composition according to embodiment 25 for use in the prevention and / or treatment of a viral infection.

[0143] Embodiment 29. A pharmaceutical composition for use according to the above embodiments, wherein the viral infection is caused by a coronavirus, preferably SARS-CoV-2.

[0144] Embodiment 30. A pharmaceutical composition according to embodiment 25 for use in the prevention and / or treatment of pathologies associated with pulmonary inflammation.

[0145] Embodiment 31. A pharmaceutical composition for use according to embodiments 28 to 30, further comprising the simultaneous or sequential administration of one or more other active substances useful for treating conditions associated with viral infections or pulmonary inflammation.

[0146] The various aspects and embodiments of the present invention are unified, benefit from, are based on, and / or are related by a common surprising finding that recombinant AAT protein produced from genetically engineered yeast can be effectively applied to the treatment of pathologies related to viral infections and / or lung inflammation.Any given feature describing one or more aspects or embodiments of the present invention can be used to describe any other aspect or embodiment of the present invention and is considered to be disclosed in that context.For example, an embodiment of the present invention relating to a method of AAT production can be considered to be related to and disclosed in the context of other aspects of the present invention, such as the AAT protein itself and / or its medical uses.

[0147] Summary of sequences disclosed in the specification: [Table 1]

[0148] Detailed Description of the Invention All cited documents, both patent and non-patent literature, are hereby incorporated by reference in their entirety.

[0149] Alpha-1 Antitrypsin (AAT): Alpha-1 antitrypsin (A1AT, AAT, PI, also known as SERPINA1) is a glycoprotein of approximately 52 kDa and one of the most abundant endogenous serine protease inhibitors (SERPIN superfamily). AAT is considered to be an acute phase protein, and AAT concentrations can increase many-fold during acute inflammation.

[0150] AAT coding region is preferably any nucleic acid encoding a naturally occurring or synthetic AAT protein sequence that exhibits AAT function with reduced, identical, similar or increased activity compared to human AAT, or functionally similar to human AAT.The amino acid sequence of AAT is available from the NCBI database under accession number 1313184B.The corresponding nucleic acid sequence encoding AAT can be provided by those skilled in the art of molecular biology or genetics.The use of sequence variants of AAT that exhibit functional analogy or similarity with the unmodified human form of AAT is encompassed by the present invention.

[0151] One AAT coding sequence (CDS) is published at http: / / www.ncbi.nlm.nih.gov / nuccore / NM_000295.4 and is one of the preferred embodiments. This sequence includes bases 262 to 1518 of the entire sequence. SEQ ID NO: 1 represents one exemplary AAT coding sequence.

[0152] In some embodiments of the invention, the CDS is codon optimized to increase protein production. The coding sequence after codon optimization preferably reads as SEQ ID NO:2.

[0153] The nucleotide sequence according to SEQ ID NO:1 and / or SEQ ID NO:2 encodes the human AAT protein of the amino acid sequence according to SEQ ID NO:3.

[0154] In an embodiment, the human AAT protein is taken according to SEQ ID NO:5, which corresponds to the human AAT sequence known in the art.

[0155] Thus, the present invention encompasses the use of a genetically modified yeast, preferably Pichia pastoris, that expresses a recombinant AAT as described herein, wherein the yeast comprises a nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence encoding an AAT protein, preferably according to SEQ ID NO: 1 or 2, such as according to SEQ ID NO: 3 or SEQ ID NO: 5, b) a nucleic acid molecule which is complementary to the nucleotide sequence according to a); c) a nucleic acid molecule comprising a nucleotide sequence having sufficient sequence identity to be functionally similar / equivalent to a nucleotide sequence according to a) or b), preferably comprising at least 70%, 75%, 80%, 85%, preferably 90%, more preferably 95% sequence identity to the nucleotide sequence according to a) or b); d) a nucleic acid molecule which, as a consequence of the genetic code, is degenerate to the nucleotide sequences according to a) to c); and / or e) A nucleic acid molecule according to the nucleotide sequence of a) to d), which has been modified by deletion, addition, substitution, translocation, inversion and / or insertion and which is functionally similar / equivalent to the nucleotide sequence of a) to d).

[0156] Thus, the present invention encompasses the use of a genetically modified yeast, preferably Pichia pastoris, as described herein, comprising a nucleotide sequence encoding an AAT protein or variant thereof according to SEQ ID NO:3 or SEQ ID NO:5, or an amino acid sequence according to SEQ ID NO:3 or SEQ ID NO:5.

[0157] The present invention encompasses further sequence variants of SEQ ID NO:3, particularly those with at least 70% sequence identity to SEQ ID NO:3 or SEQ ID NO:5, preferably at least 75%, 80%, 85%, 90%, or at least 95% sequence identity to SEQ ID NO:3 or SEQ ID NO:5. Such sequence variants are preferably functionally similar or equivalent to the human AAT disclosed herein. Variations in protein length are also encompassed by the present invention, provided that the functional equivalence of the human AAT of SEQ ID NO:3 or SEQ ID NO:5 is maintained. For example, truncation or extension of the protein length of up to 50 amino acids, 40 amino acids, 30 amino acids, 20 amino acids, or 10 amino acids may maintain AAT activity and are therefore encompassed by the present invention.

[0158] Functionally similar sequences refer to the ability to encode functional AAT gene products and to achieve the same or similar functional effects as human AAT. The function of AAT can be determined by its ability to inhibit various proteases, such as trypsin, in vitro, or its ability to inhibit neutrophil elastase (described below). Suitable assays for determining protease activity or for determining neutrophil elastase activity are known to those skilled in the art.

[0159] Also included within the scope of the present invention are protein modifications of AAT proteins that may occur by substitutions in amino acid sequences and nucleic acid sequences that code for such molecules. Substitutions, as defined herein, are modifications made to the amino acid sequence of a protein, whereby one or more amino acids are replaced with the same number (various) amino acids to generate a protein that contains an amino acid sequence that differs from the original protein. In some embodiments, this modification will not significantly change the function of the protein. As with additions, substitutions may be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly changing the function of a protein. This is especially true when the modification concerns "conservative" amino acid substitutions, which are the replacement of one amino acid with another amino acid of similar properties. Such "conserved" amino acids may be natural or synthetic amino acids that, due to size, charge, polarity and conformation, can be substituted without significantly affecting the structure and function of the protein. Often, many amino acids can be substituted by conservative amino acids without adversely affecting the function of the protein. In general, the non-polar amino acids Gly, Ala, Val, Ile and Leu, the non-polar aromatic amino acids Phe, Trp and Tyr, the neutral polar amino acids Ser, Thr, Cys, Gln, Asn and Met, the positively charged amino acids Lys, Arg and His, and the negatively charged amino acids Asp and Glu represent groups of conservative amino acids. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can be substituted for each other, even though they belong to different groups.

[0160] The term "conservative amino acid substitution" is well known in the art and refers to the replacement of a particular amino acid with one that has similar properties (e.g., similar charge or hydrophobicity, similar bulkiness). Examples include aspartic acid for glutamic acid, or isoleucine for leucine. A conservative substitution variant 1) has only conservative amino acid substitutions with respect to the parent sequence, 2) has at least 90% sequence identity with respect to the parent sequence, preferably at least 95% identity, 96% identity, 97% identity, 98% identity, or 99% or more identity, and 3) retains neuroprotective or neurorepair activity. In this regard, any conservative substitution variant of the above-mentioned polypeptide sequence is contemplated according to the present invention. Such variants are considered to be "AAT proteins".

[0161] As used herein, "percent (%) sequence identity" with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent of sequence identity, and does not consider conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity or percent nucleic acid sequence identity can be accomplished in a variety of ways that are within the scope of those skilled in the art, for example, using publicly available computer software programs. Software such as BLAST or Clustal allows for such alignment of sequences and calculation of percent identity. As used herein, the percent homology between two sequences is equal to the percent identity between the sequences. Determination of percent identity or homology between sequences can be performed, for example, using the GAP program (Genetics Computer Group, software; currently available through Accelrys at http: / / www.accelrys.com), and alignment can be performed, for example, using the ClustalW algorithm (VNTI software, InforMax Inc.). Sequence databases can be searched using the nucleic acid sequence of interest. Database search algorithms are typically based on the BLAST software (Altschul et al., 1990). In some embodiments, the percent homology or identity can be determined along the entire length of the nucleic acid.

[0162] In embodiments, the AAT may include 0-10 amino acid additions or deletions at the N-terminus and / or C-terminus of the relevant sequence, meaning that the polypeptide may a) have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acids at its N-terminus and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted at its C-terminus, or b) have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acids at its C-terminus and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides deleted at its N-terminus. or c) may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at its N-terminus and may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at its N-terminus; or d) may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted at the N-terminus and may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted at its C-terminus.

[0163] Further, in addition to the polypeptides described herein, peptide mimetics are also contemplated. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. These types of non-peptide compounds are called "peptide mimetics" or "peptidomimetics" (Fauchere (1986) Adv. Drug Res. 15:29, Veber and Freidinger (1985) TINS ​​p. 392, and Evans et al. (1987) J. Med. Chem. 30:1229) and are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce the same therapeutic or prophylactic effect. In some embodiments, it may be preferable to use a peptide mimetic to extend the stability of the polypeptide when administered to a subject. To this end, a peptide mimetic of the polypeptide that is not cleaved by the human proteasome may be preferred.

[0164] The nucleic acid molecule encoding the AAT of the present invention can be codon-optimized according to standard methods in the art for expression in cells containing the target DNA of interest.For example, when the intended target nucleic acid is in yeast cells, the yeast codon-optimized polynucleotide encoding the AAT is intended for use in the construct described herein.

[0165] In other embodiments, alpha-1 antitrypsin (AAT) protein may be replaced by other serine protease inhibitors. Thus, any features related to AAT as disclosed herein may be related to other serine protease inhibitors. In one embodiment, the serine protease inhibitor is a trypsin-like serine protease inhibitor.

[0166] Trypsin-like serine protease inhibitors inhibit serine proteases that have trypsin-like activity. Serine serves as the nucleophilic amino acid at the enzymatic active site of serine protease enzymes. A number of trypsin-like serine proteases are being actively pursued as therapeutic targets for inhibition. Viral entry by endocytosis depends on target cell proteases, such as serine proteases.

[0167] A viral infection is target cell serine protease dependent when the virus requires a serine protease of the target cell for viral entry and proliferation.

[0168] Serine proteases in target cells activate viral spike proteins required for fusion of the virus with the target cell membrane and release of the viral genome into the cytoplasm of the target cell. Targeting host cell serine proteases can prevent viral proliferation of endocytosis-dependent viruses, and thus inhibiting target cell serine proteases is an effective method for treating subjects infected with endocytosis-dependent viruses. Serine protease inhibitors, preferably trypsin-like serine protease inhibitors, inhibit viral infection and proliferation. Non-limiting examples of trypsin-like serine protease inhibitors include camostat, aprotinin, benzamidine, gabexate, leupeptin, nafamostat, pepstatin A, ribavirin, sepimostat, urinastatin, and patamostat.

[0169] molecule: As used herein, "nucleic acid" or "nucleic acid molecule" is meant to refer to a molecule composed of a chain of monomeric nucleotides, such as, for example, a DNA molecule (e.g., cDNA or genomic DNA). The nucleic acid may, for example, code for a promoter, an AAT gene or a portion thereof, or a regulatory element. The nucleic acid molecule may be single-stranded or double-stranded.

[0170] "AAT nucleic acid" refers to a nucleic acid containing the AAT gene or a part thereof, or a functional variant of the AAT gene or a part thereof. Examples of functional variants of genes include silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or variants of genes that do not significantly change gene function, such as minor mutations such as deletions.

[0171] As used herein, the term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, isolated from a naturally occurring gene or modified or synthesized to contain a segment of nucleic acid in a manner not found in nature. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present disclosure.

[0172] A DNA sequence that "encodes" a particular AAT protein (including fragments and portions thereof) is a nucleic acid sequence that is transcribed into a particular RNA and / or protein. The DNA polynucleotide may encode an RNA (mRNA) that is translated into a protein, or the DNA polynucleotide may encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or DNA target RNA; also called "non-coding" RNA or "ncRNA").

[0173] As used herein, the term "gene" or "coding sequence" is meant to broadly refer to a DNA region that codes for a protein (transcribed region). A coding sequence, when placed under the control of an appropriate regulatory region, such as a promoter, is transcribed (DNA) and translated (RNA) into a polypeptide. A gene may include several operably linked fragments, such as a promoter, a 5'-leader sequence, a coding sequence, and a 3'-untranslated sequence, including a polyadenylation site. The phrase "expression of a gene" refers to the process by which a gene is transcribed into RNA and / or translated into an active protein.

[0174] As used herein, "protein" or "polypeptide" refers to both peptides and proteins. In the present invention, the polypeptides may be naturally occurring or recombinant (i.e., produced by recombinant DNA technology) and may contain mutations (e.g., point mutations, insertion mutations and deletion mutations) and other covalent modifications (e.g., glycosylation and labeling (with biotin, streptavidin, fluorescein and radioisotopes) or other molecular linkages to additional components). For example, PEGylated proteins are included within the scope of the present invention. PEGylation has been widely used as a post-production modification method to improve the biomedical efficacy and physicochemical properties of therapeutic proteins. The applicability and safety of this technology have been proven over the years with various PEGylated pharmaceuticals (see Jevsevar et al, Biotechnol J. 2010 Jan;5(1):113-28). In some embodiments, the polypeptides described herein are modified to exhibit longer in vivo half-life and resist degradation compared to unmodified polypeptides. Such modifications, such as cyclized polypeptides, polypeptides fused to vitamin B12, stapled peptides, protein lipidization, and substitution of natural L-amino acids with D-amino acids, are known to those of skill in the art (see Bruno et al, Ther Deliv. 2013 Nov; 4(11): 1443-1467).

[0175] Production of recombinant AAT in yeast The present invention relates to a method for producing recombinant AAT or a fragment(s) thereof from genetically modified yeast, comprising culturing a genetically modified yeast comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination, expressing the recombinant AAT in the cultured yeast, and isolating the recombinant AAT from the culture.

[0176] Yeast expression platforms that can be used in the context of the present invention include strains of yeast that are used to produce large amounts of proteins, here AAT, for research or industrial applications. Although yeast often consumes more resources to maintain than, for example, bacteria, it is necessary to use a yeast expression platform because certain products can only be produced by eukaryotic cells such as yeast. Yeasts differ in their productivity and ability to secrete, process and modify proteins. Therefore, different types of yeast (i.e., different expression platforms) are more suitable for different research and industrial applications. Importantly, expression and production of proteins in yeast is advantageous because yeast can grow rapidly in large containers, produce proteins in an efficient manner, and produce and modify protein products that are safe and ready for human consumption in the context of cosmetic compositions.

[0177] The production of recombinant therapeutic agents is a rapidly developing field within therapeutic medicine. Yeast is an established eukaryotic host for heterologous protein production and offers unique advantages in the synthesis of pharmaceutical recombinants. Yeast is adept at vigorous growth on inexpensive media, is easily genetically manipulated, and allows for eukaryotic post-translational modifications. A number of yeasts, including Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Alcustura adeninivorans, Kluyveromyces lactis, and Schizosaccharomyces pombe, are exemplary yeast hosts for recombinant protein production and may be employed accordingly.

[0178] Yeast is a common host for producing proteins from recombinant DNA. Yeast offers relatively easy genetic manipulation, and rapid growth to high cell densities in inexpensive media. As a eukaryote, yeast can perform protein modifications such as glycosylation, which are common in eukaryotic cells but relatively rare in bacteria. Because of this, yeast can produce complex proteins that are identical or very similar to mammalian, especially human, natural products. The most commonly used yeast expression platform is based on baker's yeast, Saccharomyces cerevisiae. However, further yeast expression platforms such as Saccharomyces pichia and especially Pichia pastoris have been investigated and are widely used for various applications based on their different properties and capabilities. For example, some of them grow on a wide range of carbon sources and are not limited to glucose as in the case of baker's yeast. Some of them are also applied in genetic engineering and production of foreign proteins and can be used in the context of the present invention.

[0179] In an embodiment, the yeast related to the present invention is from the family Saccharomycetaceae, preferably Saccharomycetaceae Pichia, more preferably Pichia pastoris.

[0180] Pichia pastoris is an excellent expression host for the production of heterologous proteins, including industrial enzymes and biopharmaceuticals. So far, this methylotrophic expression system has been successfully utilized in the production of numerous recombinant proteins, including human erythropoietin, phospholipase C, phytase, human superoxide dismutase, trypsin, human serum albumin, collagen, and human monoclonal antibody 3H6 Fab fragment. Compared to other yeast species, P. pastoris is more efficient in the secretory production of recombinant proteins. The industrial interest of this host is also due to the strong methanol-regulated alcohol oxidase promoter (AOX1), highly efficient secretion mechanism, post-translational modification capability, and high cell density growth on defined media. A variety of recombinant proteins have been expressed using P. pastoris. There have been several successful examples of therapeutic protein production in P. pastoris. A previous study (2016) demonstrated the production of nanobodies (VHHs) against Clostridium botulinum neurotoxin type E (BoNT / E) in P. pastoris. A product yield of 16 mg / l was achieved, exceeding the production level by E. coli. Furthermore, Xia et al. showed the production of 30 mg / l of recombinant angiogenin in this yeast. Furthermore, productivities of 111 mg / l of human adiponectin, 8.1 g / l of recombinant xylanase, and 260 mg / l of anti-HIV antibodies have been reported in P. pastoris.

[0181] The average concentration of AAT in plasma is 1.3 mg / ml, with a half-life of 3-5 days. Protein size, glycosylation pattern, metastable inhibitory properties of AAT, and production costs represent challenges in recombinant AAT production. The methylotrophic yeast P. pastoris is an attractive host for human AAT production. P. pastoris has the capacity to introduce many of the post-translational modifications such as glycosylation and proteolytic processing that result from trafficking in the secretory pathway. For glycoproteins such as AAT, these modifications are crucial for proper function and / or structure.

[0182] In an embodiment, AAT is expressed in P. pastoris as a fusion protein to a histidine tag (His tag) that facilitates the purification process. The signal sequence from Saccharomyces cerevisiae, the α-factor secretion signal, was included to direct secretion of the protein into the extracellular medium. This robust expression system utilized the highly inducible alcohol oxidase 1 (AOX1) promoter to express large amounts of glycosylated proteins. P. pastoris produced AAT activity, which was characterized using an elastase inhibition assay, respectively.

[0183] In embodiments, the genetically modified yeast is of the Saccharomycete family, preferably Saccharomycetaceae saccharomyces, more preferably Saccharomyces cerevisiae. In embodiments, S. cerevisiae may be a preferred host over bacteria. The S. cerevisiae expression system is one of the most commonly used eukaryotic organisms and has been utilized to study various biological phenomena and as a model for recombinant production of therapeutic proteins. Potential issues in protein glycosylation with S. cerevisiae have been addressed, making S. cerevisiae a viable yeast for therapeutic protein production. For example, disruption of Mnn2p and Mnn11p genes associated with glycosylation modification pathways has been shown to improve the production of recombinant cellulases. Furthermore, removal of the α-1,6-mannosyltransferase Och1p enhanced the production of active human tissue-type plasminogen activator. These studies showed that N-glycosylation modifications also lead to increased protein secretion. Recombinant proteins can be expressed intracellularly or directed to the secretion apparatus using secretion signal peptides. A frequently used signal sequence that functions in all yeast expression systems is the prepro sequence of mating factor alpha 1 (MFalpha1).

[0184] Saccharomyces cerevisiae also has several important advantages from a safety standpoint, which encourages the use of this system in various industrial processes. S. cerevisiae is generally regarded as safe (GRAS) since it is non-pathogenic and has been used historically in various nutritional industries and in the production of biopharmaceuticals. On the other hand, current knowledge of yeast genetics, physiology, and fermentation facilitates the use of this organism in the production of useful products. Hepatitis B surface antigen, hirudin, insulin, glucagon, urate oxidase, macrophage colony-stimulating factor, and platelet-derived growth factor are examples of S. cerevisiae-derived products on the market. Alternative expression systems, including the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha, and the non-methylotrophic yeasts Yarrowia lipolytica, Kluyveromyces lactis, and Alcusla adeninivorans, are also viable alternatives.

[0185] Post-translational modifications In one embodiment, the methods used herein involve post-translational modification of recombinant AAT protein.

[0186] In embodiments, the post-translational modification is O-glycosylation, N-glycosylation, N-terminal methionine removal, N-acetylation and / or phosphorylation, or any combination thereof.

[0187] In embodiments, the recombinant AAT protein has one or more human-like glycoform patterns.

[0188] In embodiments, the methods used herein involve modifying recombinant AAT protein in vitro after isolation from yeast.

[0189] In some embodiments, the modification is the covalent attachment of the recombinant AAT protein to a biocompatible polymer.

[0190] The term "post-translational modification" refers to any change in the translated polypeptide chain of AAT that occurs after or during translation. This includes modifications of the amino acid side chains of the polypeptide or modifications of the terminal amino or carboxyl groups. Post-translational modification refers to the attachment of biochemical groups, such as acetate moieties, phosphate moieties, carbohydrate moieties, lipids, etc., to amino acid side chains to alter the biochemical and physical properties of a protein after translation. Many proteins undergo post-translational modifications shortly after translation, some after protein folding, and some after localization. The most common post-translational modifications of proteins are phosphorylation, glycosylation, methylation, ubiquitination, S-nitrosylation, and N-acetylation.

[0191] Such modifications may be covalent modifications enzymatically performed by the expression system following protein biosynthesis.Post-translational modifications further include enzymatic cleavage of peptide bonds and processing of translated proteins, covalent addition of specific chemical groups, polymers, lipids, carbohydrates, or even entire proteins to amino acid side chains.These chemical modifications of polypeptide chains after biosynthesis broaden the range of amino acid structures and properties, thus diversifying the structure and function of proteins.In a preferred embodiment, recombinant AAT protein undergoes post-translational modifications such as phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and proteolysis.

[0192] In one embodiment of the invention, the secreted or released AAT protein undergoes glycosylation, encompassing a diverse selection of sugar moieties added to proteins, ranging from simple monosaccharide modifications of nuclear transcription factors to highly complex branched polysaccharide modifications of cell surface receptors in yeast expression systems, preferably O-glycosylation and / or N-glycosylation. N-glycosylation is the attachment of carbohydrate to asparagine of a polypeptide. O-glycosylation is the attachment of carbohydrate to serine / threonine.

[0193] In an embodiment, the modification of recombinant AAT protein is the covalent attachment of recombinant AAT to a biocompatible polymer such as polyethylene glycol (PEG). As used herein, the term "PEGylation" refers to the process of both covalent and non-covalent attachment of polyethylene glycol polymer chains to molecules and macrostructures, such as drugs, or bioactive proteins or vesicles. PEGylation is routinely performed by incubating a reactive derivative of PEG with a target molecule. The covalent attachment of PEG to a protein "masks" the agent from the host's immune system (reducing immunogenicity and antigenicity), increases its hydrodynamic size (size in solution), and extends circulation time by reducing renal clearance. In one aspect, PEGylation can occur at the N-terminus of recombinant AAT protein.

[0194] As used herein, the term "biocompatible polymer" refers to a polymer that is compatible with exposure to the body and body fluids. Biocompatible polymers are both synthetic and natural polymers that support the immediate surroundings of biological systems or work intimately with living cells. They are used to measure, treat, enhance, or replace any tissue, organ, or function of the body. Biocompatible polymers improve bodily functions without altering normal function and without causing allergic or other side effects. This includes advances in tissue culture, tissue scaffolds, transplants, artificial grafts, wound fabrication, controlled drug delivery, bone filling materials, and the like. Biocompatible polymers are, for example, polystyrene (PS), polypropylene (PP), polyvinyl chloride (PVC), polyethylene (PE), polyurethane (PU), polycarbonate (PC), polyethylene terephthalate (PET), polyether ether ketone (PEEK), and the like.

[0195] As used herein, "polyethylene glycol" is a polyether compound that has many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide or polyoxyethylene, depending on its molecular weight.

[0196] In certain embodiments, recombinant AAT protein is conjugated to water-soluble polymer.This can be produced by any of various chemical methods known in the art.For example, in one embodiment, recombinant AAT protein is modified by conjugating PEG to the free amino group of protein using N-hydroxysuccinimide (NHS) ester.In another embodiment, water-soluble polymer, such as PEG, is coupled to free SH group using maleimide chemistry, or by coupling PEG hydrazide or PEG amine to carbohydrate moiety of recombinant AAT protein after pre-oxidation.

[0197] According to one embodiment of the invention, PEGylation is performed using PEG of 20 kDa or greater, which is conjugated to AAT to provide a wrapping effect around the protein, protecting it against loss after binding to scavenger receptors or degradation by inactivating proteases.

[0198] PEG is a polymer in the form of a linear or branched chain, and PEG with a small molecular weight cannot completely encapsulate the protein, and therefore cannot completely protect the protein.Therefore, the molecular weight must be equal to or greater than a critical level to fully encapsulate the protein to be protected.The PEGylation of the recombinant AAT protein of the present invention can be carried out by substituting the amino acid at the PEGylation position with cysteine, and then conjugating it to PEG that contains a cysteine-specific acryloyl group, sulfone group or maleimide group at one end.

[0199] Glycosylated AAT produced by yeast In embodiments, the post-translational modification is O-glycosylation, N-glycosylation, N-terminal methionine removal, N-acetylation and / or phosphorylation, or any combination thereof. In embodiments, the recombinant AAT protein has one or more human-like glycoform patterns.

[0200] As used herein, the yeast for producing AAT allows AAT to be modified into a human-like structure. The term "human-like" structure or "human-like" glycoform pattern refers to a glycosylation pattern that, for example, extends the serum half-life of recombinant AAT and / or stability comparable to human plasma-derived AAT. The "human-like" glycoform pattern is preferably different from but similar to the human glycosylation pattern, to the extent that it extends the serum half-life of recombinant AAT compared to unmodified AAT protein. The "human-like" glycoform pattern may, for example, include one or more glycans selected from the group consisting of mannose, N-acetylglucosamine (GlcNAc), galactose, N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid, and xylose.

[0201] The glycosylation pattern of recombinant AAT produced by yeast and other post-translational modifications by yeast produce a new type of recombinant AAT, which can be verified by a person skilled in the art by SDS-page analysis. The shift of the band up or down by recombinant AAT produced from yeast such as Pichia pastoris compared to the band of AAT from plasma can be a clear example of a new type of recombinant AAT.

[0202] Isolation / purification of recombinant AAT: According to the invention, AAT is preferably purified or isolated from culture supernatants or other preparations obtained, for example, from yeast cultures, using means known to those skilled in the art. Recovery of the material obtained by fermentation of Pichia pastoris is possible using standard laboratory equipment to process the product from the supernatant. For example, a suitable process includes a centrifugation step and / or depth filtration, optionally followed by (sterile) membrane filtration. Also available is low-speed centrifugation sufficient to pellet the yeast cells and leave a clear, transparent supernatant, which may also be evidence of the absence of bacterial contamination. Such a procedure results in a supernatant ready for subsequent purification steps, such as chromatography.

[0203] For example, various methods, including liquid chromatography, suitable for separating the protein of interest can be selected by those skilled in the art without undue effort. For example, the solid phase used in chromatography, known as the chromatographic medium or resin, is typically an engineered porous inert support functionalized with various chemical groups that determine the interaction with the molecules to be separated. Commonly used separation modes applicable to the rAAT described herein are based on, but are not limited to, specific binding interactions (affinity chromatography), charge (ion exchange chromatography), size (size exclusion chromatography / gel filtration chromatography), hydrophobic surface area (hydrophobic interaction chromatography and reversed phase chromatography), and / or multiple properties (multimodal or mixed mode chromatography).

[0204] Medical Indications: In one aspect of the invention there is provided a pharmaceutical composition or combination as described herein for use in the treatment and / or prevention of a viral infection and / or a condition associated with a viral infection in a subject. Preferred viral infections or associated diseases to be treated are those described herein.

[0205] As used herein, "viral infection" occurs when a pathogenic virus invades the body of an organism.The type of virus is preferably selected from the group consisting of adenovirus, coxsackievirus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human herpes virus type 8, HIV, RSV, influenza virus, measles virus, mumps virus, human papillomavirus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, varicella zoster virus, and SARS-CoV-2 virus.

[0206] In one embodiment, the viral family is preferably selected from the group of Adenoviridae, Picornaviridae, Herpesviridae, Picornaviridae, Hepadnaviridae, Flaviviridae, Herpesviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papillomaviridae, Picornaviridae, Laboviriidae, Togaviridae, Herpesviridae and Coronaviridae.

[0207] By way of example, respiratory viral diseases may occur due to infection with adenovirus, coronavirus (common cold virus), influenza virus, parainfluenza virus, parvovirus B19 (fifth disease), respiratory syncytial virus (RSV), or rhinovirus (common cold). Additional viral respiratory diseases or viruses may be associated with HBoV, human bocavirus; HCoV, human coronavirus; HMPV, human metapneumovirus; HPIV, human parainfluenza virus; HRSV, human respiratory syncytial virus; HRV, human rhinovirus; PCF, pharyngoconjunctival fever; SARS, severe acute respiratory syndrome; SARS-CoV, SARS-associated coronavirus; URI, upper respiratory tract infection.

[0208] Any one or more of the aforementioned viruses may be susceptible to the inventive treatment described herein, and treatment of each virus, in itself, represents an embodiment of the invention.

[0209] In an embodiment, the present invention also relates to a recombinant alpha 1-antithricin (AAT) protein or fragment thereof expressed in the genetically modified yeast for use in the treatment or prevention of one or more of the above mentioned viral infections.

[0210] In an embodiment, the present invention also relates to a recombinant alpha 1-antithricin (AAT) protein or a fragment thereof expressed in genetically modified yeast for use in the treatment or prevention of influenza infection.

[0211] In one aspect of the invention there is provided a pharmaceutical composition or combination according to the invention as described herein for use in the treatment of a condition associated with a coronavirus, such as the SARS-coronavirus, which condition is preferably COVID-19 or a SARS-coronavirus associated respiratory disease.

[0212] As used herein, a "patient" or "subject" may be a vertebrate. In the context of the present invention, the term "subject" includes both humans and animals, particularly mammals, and other organisms.

[0213] As used herein, "subject in need thereof" refers to a subject suffering from or at risk of developing a disease, particularly a coronavirus disease. A subject in need thereof may be a subject hospitalized with symptoms of coronavirus disease, a subject with symptoms of coronavirus disease and in an outpatient setting or at home, or a subject with asymptomatic coronavirus disease and in an outpatient setting or at home.

[0214] In the present invention, "treatment" or "therapy" generally means obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, considering that the disease and / or condition is completely or partially prevented, for example by reducing the subject's risk of having the disease or condition, or may be therapeutic, considering that the disease and / or adverse effects of the disease are partially or completely cured.

[0215] In the present invention, "therapy" includes any treatment of a disease or condition in a mammal, particularly a human, such as the following treatments (a) to (c): (a) preventing the onset of the disease, condition or symptom in a patient; (b) arresting the symptoms of the condition, i.e. preventing the progression of the symptom; (c) ameliorating the symptoms of the condition, i.e. inducing regression of the disease or condition.

[0216] In one embodiment, the treatment described herein relates to either reducing or inhibiting coronavirus infection or symptoms thereof by preventing viral entry into target cells. The prophylactic treatment described herein is intended to encompass prevention or reduction of risk of coronavirus infection by reducing the likelihood of coronavirus infection of cells via interaction with ACE2 protein following treatment with an agent described herein.

[0217] As used herein, a "patient with symptoms of infectious disease" is a subject who exhibits one or more of the following symptoms, including but not limited to: fever, diarrhea, fatigue, muscle pain, cough, animal bites, difficulty in breathing, severe headache with fever, rash or swelling, unexplained or prolonged fever or vision problems. Other symptoms may include fever and chills, extremely low body temperature, decreased urine output (oliguria), rapid pulse, shortness of breath, nausea and vomiting. In a preferred embodiment, the symptoms of infectious disease are fever, diarrhea, fatigue, muscle pain, rapid pulse, shortness of breath, nausea and vomiting, and / or cough.

[0218] As used herein, a patient having "symptoms of a respiratory tract viral infection" is a subject who exhibits one or more cold-like symptoms or flu-like illness, including, but not limited to, fever, cough, runny nose, sneezing, sore throat, difficulty breathing, headache, muscle aches, fatigue, rapid pulse, rapid breathing, nausea and vomiting, loss of taste and / or smell, and / or fatigue (feeling unwell).

[0219] In some embodiments, symptoms of infection with a SARS-virus are fever, sore throat, cough, muscle aches or fatigue, and in some embodiments further include sputum production, headache, hemoptysis and / or diarrhea. In some embodiments, symptoms of infection with a SARS-coronavirus, such as SARS-CoV-2, are fever, sore throat, cough, loss of taste and / or smell, shortness of breath and / or fatigue.

[0220] As used herein, the term "patient at risk of developing Severe Acute Respiratory Syndrome (SARS)" relates to a subject, preferably at an increased (e.g., above average) risk of developing SARS apart from any given person in the general population. In some embodiments, the patient has symptoms of SARS or symptoms of SARS coronavirus infection. In some embodiments, the patient does not have symptoms of SARS or symptoms of SARS coronavirus infection. In some embodiments, the subject has been in contact with people with SARS coronavirus infection or symptoms. In some embodiments, a person at risk of developing SARS has been tested for the presence of SARS coronavirus infection. In some embodiments, a person at risk of developing SARS has tested positive for the presence of SARS coronavirus infection, preferably coronavirus infection.

[0221] In an embodiment, a patient at risk of developing SARS is an asymptomatic patient who does not (yet) show specific symptoms of SARS. An asymptomatic patient may be at risk of developing SARS because the patient has been in contact with a person infected with SARS coronavirus. For example, an asymptomatic patient may have been identified as at risk of developing SARS by a software application (app) installed on a smartphone or a corresponding (mobile) device, indicating physical proximity or short physical distance with an infected patient using the corresponding app on the respective mobile device / smartphone. Other methods of determining contact / physical proximity to an infected person are known to the skilled person and apply equally to the method of the present invention.

[0222] In some embodiments, the patient suffering from or at risk of developing Severe Acute Respiratory Syndrome (SARS) suffers from a coronavirus infection.

[0223] Coronaviruses are a group of related viruses that cause disease in mammals and birds. The scientific name for coronaviruses is the Orthocoronavirus or Coronavirinae subfamily. Coronaviruses belong to the Coronaviridae family. This family is divided into the Coronavirinae and Torovirinae subfamily, which are further divided into six genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, Deltacoronavirus, Torovirus, and Buffinivirus. Viruses in the Alphacoronavirus and Betacoronavirus genera primarily infect mammals, while Gammacoronaviruses infect birds, and members of the Deltacoronavirus genus are found in both mammalian and avian hosts.

[0224] In humans, coronaviruses cause respiratory tract infections that can be mild, such as the common cold, and potentially fatal, such as SARS, MERS, and COVID-19. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid with helical symmetry. The genome size of coronaviruses ranges from approximately 27 kilobases to 34 kilobases, making them the largest of the known RNA viruses.

[0225] Various species of human coronaviruses are known, including, but not limited to, human coronavirus OC43 (HCoV-OC43) of the genus β-CoV, human coronavirus HKU1 (HCoV-HKU1) of the genus β-CoV, human coronavirus 229E (HCoV-229E), α-CoV, human coronavirus NL63 (HCoV-NL63), α-CoV, Middle East Respiratory Syndrome-related Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0226] Coronaviruses vary widely in their risk factors. Some (e.g. MERS-CoV) can kill over 30% of those they infect, while others are relatively harmless, like the common cold. Coronaviruses cause the common cold, with major symptoms such as fever and sore throat, e.g. due to swelling of the pharyngeal tonsils, mainly in winter and early spring. Coronaviruses can cause pneumonia (either direct viral pneumonia or secondary bacterial pneumonia) and bronchitis (either direct viral bronchitis or secondary bacterial bronchitis). Coronaviruses can also cause SARS.

[0227] Advances in nucleic acid sequencing technology (commonly referred to as next-generation sequencing, NGS) have provided a large amount of sequence data from a variety of biological samples, allowing the characterization of both known and novel viral strains, and therefore established methods for determining coronavirus infections are available.

[0228] As used herein, "Long COVID" is also known as Post-COVID-19 Syndrome, Post-Acute Sequelae of COVID-19 (PASC), or Chronic COVID Syndrome (CCS), and refers to a condition characterized by long-term sequelae that emerge or persist after the typical recovery period of COVID-19. Long COVID can affect almost every organ system with sequelae including respiratory disorders, neurological and neurocognitive disorders, mental health disorders, metabolic disorders, cardiovascular disorders, gastrointestinal disorders, malaise, fatigue, musculoskeletal pain, and anemia. A variety of symptoms are commonly reported, including fatigue, headache, shortness of breath, anosmia (loss of smell), parosmia (distortion of smell), muscle weakness, low-grade fever, and cognitive impairment.

[0229] Other symptoms or conditions associated with Long COVID include, but are not limited to, extreme fatigue, persistent cough, muscle weakness, low-grade fever, difficulty concentrating (brain fog), memory loss, dysphoria, sleep disorders sometimes accompanied by depression and other mental health issues, headaches, joint pain, needle pains in the arms and legs, bouts of diarrhea and vomiting, loss of taste and smell, sore throat and difficulty swallowing, new onset of diabetes and high blood pressure, heartburn (gastroesophageal reflux disease), skin rash, shortness of breath, chest pain, palpitations, kidney problems (acute kidney injury and chronic kidney disease), changes in oral health (teeth, saliva, gums), anosmia (lack of smell), parosmia (changes in smell), tinnitus, blood clotting (deep vein thrombosis and pulmonary embolism).

[0230] In one embodiment, long COVID, for new or ongoing symptoms more than four weeks after the onset of acute COVID-19, is divided into two other categories: ongoing symptomatic COVID-19, for effects 4-12 weeks after onset, and post-COVID-19 syndrome, for effects lasting more than 12 weeks after onset.

[0231] In other embodiments, Long COVID symptoms in individuals who "do not fully recover for weeks" are collectively referred to as Post-Acute Sequelae of SARS-CoV-2 Infection (PASC). Long COVID symptoms include fatigue, shortness of breath, "brain fog," sleep problems, intermittent fever, gastrointestinal symptoms, anxiety, and depression. Symptoms can last for months and range from mild to incapacitating, with new symptoms appearing long after the time of infection. The CDC's term Post-Covid Conditions qualifies Long Covid as symptoms present more than four weeks after initial infection.

[0232] Composition and Administration: The AAT protein or compounds containing the protein described herein can include various types of carriers depending on whether they are administered in solid, liquid, or aerosol form, and whether the route of administration, such as an injection, requires sterility.

[0233] The active agent AAT can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, intramucosally, intrapericardially, subumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, directly, via a catheter, via lavage, in a cream, in a lipid composition (e.g., liposomes), topically applied by sponge, or by other methods known to those skilled in the art or any combination of the above (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0234] The present invention encompasses the treatment of a patient by introducing a therapeutically effective number of polypeptides into the subject or into the subject's bloodstream. As used herein, "introducing" a polypeptide into the subject's bloodstream includes, but is not limited to, introducing said polypeptide into one of the subject's veins or arteries via injection. Such administration can be performed, for example, once, multiple times, and / or over an extended period of time, such as one or more times. A single injection is preferred, but in some instances multiple injections over time (e.g., four times a year, twice a year, or once a year) may be necessary. Such administration is also preferably performed using a mixture of the polypeptide and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are known to those of skill in the art and include, but are not limited to, 0.01 M to 0.1 M, preferably 0.05 M, phosphate buffer or 0.8% saline.

[0235] As used herein, the term "therapeutically effective amount" is interchangeable with either "therapeutically effective dose" or "sufficient / effective amount or dose" and refers to a dose that produces the required therapeutic effect. Specifically, an effective dose generally refers to an amount of a composition disclosed herein sufficient to induce immunity, prevent and / or ameliorate coronavirus infection, or reduce at least one symptom associated with coronavirus infection, and / or enhance the effectiveness of another therapeutic composition. An effective dose may refer to an amount of a composition sufficient to delay or minimize the onset of an infection. An effective dose may refer to an amount of a composition sufficient to prevent infection with a virus or reduce the risk of infection with a virus. An effective dose may also refer to an amount of a composition that provides a therapeutic benefit in the treatment or management of an infection. In addition, an effective dose may be an amount for a composition alone or in combination with other therapies that provides a therapeutic benefit in the treatment or management of a viral infection. An effective dose may be an amount sufficient to enhance a subject's (particularly a human's) own immune response to subsequent exposure to coronavirus. The exact effective dose depends on the purpose of the treatment and can be ascertained by one of skill in the art using known techniques.

[0236] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0237] Additionally, such pharma-ceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions and emulsions, most preferably aqueous solutions. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions and suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as Ringer's dextrose, Ringer's dextrose-based, and the like. Fluids commonly used for intravenous administration can be found, for example, in Remington: The Science and Practice of Pharmacy, 20th Ed., p. 808, Lippincott Williams S-Wilkins (2000). Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, inert gases, and the like.

[0238] The term "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution or suspension in liquid prior to injection can also be prepared. The preparation can also be emulsified.

[0239] Examples of parenteral dosage forms include aqueous solutions of the active substance, isotonic saline, 5% glucose, or other well-known pharma- ceutically acceptable liquid carriers, such as liquid alcohols, glycols, esters, and amides. A parenteral dosage form according to the present invention may be in the form of a reconstitutable lyophilizate containing a dose of a composition comprising granulin. In one aspect of this embodiment, any of a number of sustained- or extended-release dosage forms known in the art may be administered, such as the biodegradable carbohydrate matrices described in U.S. Pat. No. 4,713,249, U.S. Pat. No. 5,266,333, and U.S. Pat. No. 5,417,982, the disclosures of which are incorporated herein by reference.

[0240] In an exemplary embodiment, a pharmaceutical formulation for general use with AAT for parenteral administration comprises: a) a pharma- ceutical active amount of granulin; b) a pharma- ceutical acceptable pH buffer providing a pH in the range of about pH 4.5 to about pH 9; c) an ionic strength modifier in a concentration range of about 0 millimolar to about 250 millimolar; and d) a water-soluble viscosity modifier in a concentration range of about 0.5% to about 7% of the total weight of the described formulation, or any combination of a), b), c), and d).

[0241] In various exemplary embodiments, pH buffers used in the compositions and methods described herein are agents known to those of skill in the art, including, for example, acetate, borate, carbonate, citrate, and phosphate buffers, as well as hydrochloric acid, sodium hydroxide, magnesium oxide, monopotassium phosphate, bicarbonate, ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid, acetic acid, disodium hydrogen phosphate, borax, boric acid, sodium hydroxide, diethylbarbituric acid, and protein, as well as various biological buffers, such as TAPS, Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES.

[0242] In another exemplary embodiment, the ionic strength adjusting agents include agents known in the art, such as glycerin, propylene glycol, mannitol, glucose, dextrose, sorbitol, sodium chloride, potassium chloride, and other electrolytes.

[0243] The compositions of the present invention are preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. The term "parenteral administration" refers to modes of administration other than into the digestive tract, such as by injection or infusion, and further refers to subcutaneous injection, intramuscular injection, or intravenous or intraperitoneal injection, which are contemplated herein. Intramuscular administration includes intravenous or intraarterial administration. Liquid pharmaceutical compositions, whether they are in solution, suspension or other similar form, may contain one or more of the following suitable excipients: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that can serve as solvents or suspending media, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Pharmaceutical compositions for injection are preferably sterile. In addition, the pharmaceutical composition to be administered may, if desired, contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0244] A single daily dose of a composition containing AAT protein can vary widely depending on the condition of the patient, the pathology being treated, the route of administration and tissue distribution of AAT, and the possibility of concomitant use of other therapeutic treatments. The effective amount of AAT administered to a patient is based on body surface area, patient weight, physician's assessment of the patient's condition, etc.

[0245] In an exemplary embodiment, an effective dose of AAT may be in the range of about 1 mg / kg patient body weight to about 500 mg / kg patient body weight, more preferably about 10 mg / kg patient body weight to about 300 mg / kg patient body weight, and even more preferably about 50 mg / kg patient body weight to about 200 mg / kg patient body weight, for example, about 100 mg / kg or about 180 mg / kg body weight.

[0246] According to the present invention, administration of the composition may occur preferably via inhalation to the mucosal surface of the subject's airway. "Inhalation administration" refers to both oral and nasal inhalation. "Oral inhalation" refers to a drug being administered by inhalation through the oral cavity, which requires the drug to be atomized into smaller droplets than drugs administered via the nasal route so that the drug can pass through the windpipe (trachea) and enter the lungs. How deep they enter the lungs depends on the size of the droplets. Smaller droplets enter deeper and greater amounts of drug are absorbed. Inside the lungs, they are absorbed into the bloodstream.

[0247] Drugs administered by this route may be delivered by a metered dose container (inhaler) in the form of a dry powder.

[0248] Alternatively, the drug can be sprayed from a solution containing the drug into a mist or into suitably sized droplets or into an aerosol by a nebulizer such as the Pari Boy Pro for inhalation.

[0249] Inhaled drugs are rapidly absorbed and act both locally and systemically. Inhalers can achieve the correct dose. Generally, only 20%-50% of the pulmonary delivery dose given in powdered particles is deposited in the lungs upon oral inhalation. The remaining 50%-70% of undeposited aerosolized particles are immediately removed from the lungs with exhalation. In an embodiment, inhaled powdered particles larger than 8 μm are structurally predisposed to deposit in the central and conducting airways by inertial impaction. In an embodiment, inhaled powdered particles with diameters between 3 μm and 8 μm tend to deposit predominantly in the transition zone of the lungs by sedimentation. In an embodiment, inhaled powdered particles with diameters less than 3 μm are structurally predisposed to deposit in the respiratory zone of the peripheral lungs primarily via diffusion.

[0250] The term "nasal inhalation" refers to a medicine or preparation, including a delivery device if applicable, whose intended site of deposition is the respiratory tract or nasal or pharyngeal area. Nasal inhalation does not include topical nasal drops and irrigation, which are deposited primarily in the nasal cavity. Drugs can also be sprayed from a solution containing the drug into a mist or into suitable sized droplets or into an aerosol by a nebulizer, such as the Pari Boy Pro for inhalation.

[0251] Simultaneous and / or sequential administration of pharmaceutical compositions by different administration routes is also contemplated in the context of the present invention. In an embodiment, the administration route of the composition comprises inhalation administration. In a preferred embodiment, parenteral administration of the pharmaceutical composition according to the present invention or the pharmaceutical composition comprising human plasma-derived AAT protein is performed simultaneously or sequentially. In another preferred embodiment, nasal and / or oral administration of the pharmaceutical composition according to the present invention or the pharmaceutical composition comprising human plasma-derived AAT protein is performed simultaneously or sequentially.

[0252] As used herein, the "airway" is generally considered to include the nose, pharynx, larynx, trachea, and lungs with their various compartments. The airways are involved in the breathing process in mammals, are part of the respiratory system, and are lined with airway mucosa or respiratory epithelium.

[0253] As used herein, a "mucosal surface" is characterized by the presence of overlying mucus, e.g., saliva, tears, nasal mucus, gastric mucus, cervical mucus and bronchial mucus, the functions of which include supplying and delivering an array of immunomodulatory and healing-promoting species, including growth factors, antimicrobial proteins and immunoglobulins.

[0254] As used herein, a "pharmaceutical excipient" can be essentially anything other than an active pharmaceutical ingredient in a composition.

[0255] In the formulation of pharmaceutical compositions, excipients are generally added along with the active pharmaceutical ingredient to: 1) protect, support or enhance the stability of the formulation; 2) bulk the formulation, in the case of potent drugs, to aid in the formulation of precise dosage forms; 3) improve patient acceptance; 4) help increase the bioavailability of the active drug; and 5) increase the overall safety and effectiveness of the formulation during storage and use.

[0256] Excipients used in the formulation of pharmaceutical compositions are subdivided into various functional classes depending on the role they are intended to play in the resulting formulation. Some excipients may have different functional roles in different formulation types, and individual excipients may have different grades, types, and sources depending on their different functional roles. Types of excipients that may be commonly used in the formulation of pharmaceutical suspensions include solvents / vehicles, cosolvents, buffers, preservatives, antioxidants, wetting agents / surfactants, antifoaming agents, flocculation modifiers, suspending agents / viscosity modifiers, flavors, sweeteners, colorants, humectants, and chelating agents.

[0257] As used herein, "cryopreservation" is a process in which organelles, cells, tissues, extracellular matrices, organs, or any other biological constructs susceptible to damage caused by unregulated chemical dynamics are preserved by cooling to very low temperatures by freezing (typically -20°C to -80°C using solid carbon dioxide, or occasionally -196°C using liquid nitrogen). At a low enough temperature, any enzymatic or chemical activity that may cause damage to the biomaterial in question is effectively stopped. Cryopreservation methods seek to reach low temperatures without causing further damage caused by the formation of ice crystals during freezing.

[0258] As used herein, an "aerosol" is a suspension of liquid and solid particles produced by an aerosol generator, such as, for example, a small volume nebulizer (SVN), a pressurized metered dose inhaler (pMDI), or a dry powder inhaler (DPI). Aerosol deposition is the process by which aerosol particles are deposited on an absorbing surface.

[0259] Aerosol devices used in clinical practice produce heterodisperse (also called polydisperse) particle sizes, meaning that a variety of sizes are present in the aerosol. Specific sizes and / or size ranges are provided in the Summary of the Invention above. Monodisperse aerosols consisting of a single particle size are rare. Polydisperse aerosols can be defined by the mass median diameter (MMD). This measure defines the particle size (in μm) above and below which 50% of the mass of the particles falls. This is the particle size that divides the mass or amount of drug evenly in the particle size distribution. This is usually given as the mass median aerodynamic diameter or MMAD, depending on how the size is measured. The higher the MMAD, the larger the diameter of the particle size.

[0260] Dry powder formulations can offer advantages over liquid formulations, such as greater stability and potentially no need for preservatives. Powders tend to adhere to the moist surface of the nasal mucosa before dissolving and being removed. The use of bioadhesive excipients or agents that slow ciliary action may decrease clearance rates and improve absorption. Many factors affect deposition and absorption, such as moisture sensitivity, solubility, particle size, particle shape, and flow properties.

[0261] According to some embodiments, the pharmaceutical composition administered via inhalation is administered once a day for at least two days. According to some embodiments, the pharmaceutical composition administered via inhalation is administered once a day for at least three days. According to some embodiments, the pharmaceutical composition administered via inhalation is administered once a day for at least four days. According to some embodiments, the pharmaceutical composition administered via inhalation is administered once a day for at least five days. According to some embodiments, the pharmaceutical composition administered via inhalation is administered once a day for at least six days. According to some embodiments, the pharmaceutical composition administered via inhalation is administered once a day for at least one week. According to some embodiments, the pharmaceutical composition administered via inhalation is administered once a day for at least eight days. According to some embodiments, the pharmaceutical composition administered via inhalation is administered once a day for at least nine days. According to some embodiments, the pharmaceutical composition administered via inhalation is administered once a day for at least ten days. According to some embodiments, the pharmaceutical composition administered via inhalation is administered once a day for ten days.

[0262] Dry powder nasal spray examples: SBNL Pharma (www.snbl.com) recently reported data from a Phase 1 trial using a zolmitriptan powder cyclodextrin formulation for absorption enhancement (μcoTM system), previously described in an in vitro study. Absorption of zolmitriptan was rapid and the relative bioavailability was higher than the commercially available tablet and nasal spray. The company has a capsule-based single-dose powder device (Fit-lizer) suitable for administration of the powder formulation according to the invention. Upon insertion into the chamber, the top and bottom of the capsule are cut off with a sharp blade. The plastic chamber is compressed by hand and, during actuation, compressed air passes through a one-way valve and the capsule, releasing the powder.

[0263] The principle of Unidose-DPTM, the Bespak (www.bespak.com), is similar to the Fit-lizer device. An air-filled compartment is compressed until a pin bursts a membrane, releasing the pressure and releasing a plume of powder. Delivery of a powder formulation of a model antibody (human IgG) has been tested in a nasal cast model based on human MRI images. Approximately 95% of the dose was delivered to the nasal cavity, but the majority of it was not deposited further than the nasal vestibule, and only about 30% was deposited in the deeper compartments of the nasal cavity.

[0264] Examples of dry powder inhalers: Astra Zeneca markets budesonide powder delivered by a Turbuhaler multi-dose inhaler modified for nasal inhalation (Rhinocort Turbuhaler™, www.az.com). It is sold in some markets as an alternative to liquid sprays for allergic rhinitis and nasal polyps.

[0265] The Aptar Group (www.aptar.com) offers a simple blister-based powder inhaler. Before use, the blister is punctured and the nosepiece of the device is placed into one nostril. The subject closes the other nostril with a finger and inhales the powder into the nose. A powder formulation of apomorphine for Parkinson's disease using this blister-based powder inhaler (BiDose™ / Prohaler™) from Pfeiffer / Aptar was in clinical development by Britannia, a UK company recently acquired by Stada Pharmaceutical (www.stada.de). Such an administration device is contemplated for administering compositions as described herein.

[0266] Examples of inhalers for liquid formulations: Inhalation solutions and suspensions are typically aqueous formulations that contain a therapeutically active ingredient and may also contain additional excipients. Aqueous oral inhalation solutions and suspensions must be sterile. Inhalation solutions and suspensions are intended to be delivered to the lungs by oral inhalation for local and / or systemic effect and are used with a designated nebulizer, e.g. Pari Boy Pro, capable of producing a flexible and variable droplet spectrum for the treatment of severe chronic respiratory diseases. Unit dose presentation is recommended for these medicines to prevent microbial contamination during use. The container closure system for these medicines consists of a container and a closure, which may be provided with a protective packaging, such as a foil overwrap.

[0267] For example, Amikacin liposomal inhalation suspension (ALIS; Arikayce™) is a liposomal formulation of the aminoglycoside antibacterial drug amikacin. The ALIS formulation is administered by inhalation after nebulization and is designed to promote targeted, localized drug delivery to the lungs while minimizing systemic exposure. Such administration devices are contemplated for administering compositions as described herein.

[0268] Examples of nasal sprays using liquid formulations: A nasal spray dispenser is typically a non-pressurized dispenser that delivers a spray containing a metered dose of the active ingredient. The dose can be metered by a spray pump or can be pre-metered during manufacture. Nasal spray units can be designed for unit dosing or can dispense up to several hundred metered sprays of a formulation containing the active pharmaceutical ingredient. Nasal sprays are applied to the nasal cavity for local and / or systemic effect.

[0269] Liquid nasal formulations are primarily aqueous solutions, although suspensions and emulsions can also be delivered. Liquid formulations are considered convenient for topical applications, especially where humidification combats the dryness and crusting that often accompanies chronic nasal disease.

[0270] For example, dispensers available from, for example, Nemera (La Verpilliere, France) or Aptar Pharma (IL, USA) are preferred. For example, Nemera offers the Advancia™ nasal spray dispenser, a high-performance pump with excellent dose consistency and prime retention, an anti-clogging actuator, no metal contact with the formulation, and a hygienic anti-actuation snap-on overcap. As a further example, AptarPharma's nasal pump technology eliminates the need for pharmaceutical companies to add preservatives to nasal spray formulations. The Advanced Preservative Free (APF) system uses tip seal and filter technology to prevent contamination of the formulation. A spring-loaded tip seal mechanism is employed with a filter membrane in the ventilation channel.

[0271] The metering and spray generating (e.g. orifice, nozzle, jet) pump mechanisms and components are used for reproducible delivery of drug formulations and these may consist of many parts of different designs precisely controlled in terms of dimensions and composition. Dispersing the formulation as a spray requires energy. This is typically achieved by forcing the formulation through a nasal actuator and its orifice. The formulation and container closure system (container, closure device, pump, and any protective packaging) collectively constitute a drug product. The design of the container closure system impacts the dosing performance of the drug product.

[0272] In some embodiments, the multi-use dispenser employs a membrane (preferably silicone) that prevents bacteria or viruses from entering the reservoir or pump device. In some embodiments, the multi-use dispenser employs a metal-free fluid path, thereby preventing oxidation of the formulation. In some embodiments, the multi-use dispenser employs a spring-loaded tip seal mechanism, thereby preventing microorganisms from entering the device during a spray event.

[0273] In one embodiment, the dispenser is configured for multiple individual spray events of between 5 μL and 1000 μL, preferably between 5 μL and 500 μL, more preferably between 10 μL and 300 μL, and even more preferably between 20 μL and 200 μL.

[0274] In one embodiment, the dispenser contains a total volume of the composition of 0.1 mL to 500 mL, preferably 1 mL to 100 mL, more preferably 2 mL to 50 mL, such as about 5 mL, 10 mL or 15 mL.

[0275] Example of a throat spray for liquid preparations: Bona (Shenzhen, China) is a supplier of medical packaging and complements. The company offers a line of long-neck sprays that provide superior dispensing and dosing, designed for throat sprays and other treatments that require standardization on the throat. The dispenser arm swings 360 degrees for easy use by the consumer. The sprayers are suitable for medicinal throat treatments as well as other treatments for topical treatments, for example, protecting the throat from viral infections. The dispensers are preferably prepared from pharmaceutical grade PP and PE, and the dispensers can be combined with any bottle size. Currently, the sprayers are available in a variety of popular sizes (18 / 410, 18 / 415, 20 / 410, 24 / 410) and can be set to dispense from 220 microliters to 50 microliters.

[0276] Simultaneous and Sequential Administration Concurrent administration of one or more active agents shall mean administration of one or more agents at the same time. For example, in one embodiment, AAT can be administered together with other active agents useful for treating a condition associated with a viral infection or pulmonary inflammation. In another embodiment, AAT can be administered by inhalation together with other administration routes including parenteral administration, including oral, nasal, intravascular, intraperitoneal, or intramuscular administration. In some embodiments, at least one administration route that is a recombinant AAT according to the present invention is required, and is preferably inhalation administration. The other administration route may alternatively be a composition comprising human plasma-derived AAT or any other recombinantly produced AAT. In an embodiment, the administration route applied simultaneously may be a sequential method.

[0277] Sequential administration of one or more active substances is intended to mean administering the therapeutic agents sequentially. In one embodiment, each therapeutic agent is administered at a different time. In another embodiment, administration of two or more therapeutic agents results in at least two therapeutic agents being administered sequentially, which is substantially simultaneous. Substantially simultaneous administration can be achieved, for example, by administering to the subject a single dose having a fixed ratio of each therapeutic agent, or by administering multiple single doses for each of the therapeutic agents. One or more therapeutic agents described herein include a pharmaceutical composition comprising at least a recombinant AAT protein according to the present invention. In an embodiment, the therapeutic agent comprises a further pharmaceutical composition comprising human plasma-derived AAT and / or another recombinantly produced AAT protein. In a preferred embodiment, the therapeutic agent comprises a further pharmaceutical composition comprising xanthohumol.

[0278] As used herein, the term "concurrently" refers to administration of one or more agents at the same time. For example, in certain embodiments, administration of AAT in combination with other active agents useful for treating conditions associated with viral infections or pulmonary inflammation is administered simultaneously. Concurrently includes administration at the same time, i.e., for the same period of time. In certain embodiments, one or more agents are administered simultaneously at the same time or on the same day. Sequential or substantially simultaneous administration of each therapeutic agent can be performed by any suitable route, including, but not limited to, oral, intravenous, subcutaneous, intramuscular, direct absorption through mucosal tissues (e.g., nose, mouth, vagina, and rectum), and ocular routes (e.g., intravitreal, intraocular, etc.). The therapeutic agents can be administered by the same route or different routes. For example, one component of a particular combination may be administered by inhalation and the other component(s) of the combination may be administered intravenously. The components may be administered in any therapeutically effective order.

[0279] Decision of simultaneous administration and sequential administration can be made according to the severity of the disease.For example, for a patient with severe symptoms of Covid-19 receiving inhalation of AAT, simultaneous and / or sequential intravenous administration of AAT protein can be decided.For example, for a Covid-19 patient, AAT is inhaled, and the AAT droplet contains xanthohumol.For example, for a Covid-19 patient receiving inhalation of AAT, another dose of xanthohumol can be administered simultaneously or sequentially.

[0280] drawing The following drawings are presented to illustrate certain embodiments of the invention without limiting its scope. [Brief description of the drawings]

[0281] [Figure 1] Vector map for recombinant protein expression in Pichia pastoris. [Diagram 2] FIG. 1. Purification by anion exchange chromatography (AEX) of supernatant containing recombinant AAT produced in Pichia pastoris. [Diagram 3] Western blot analysis of recombinant AAT fractions obtained from anion exchange chromatography (AEX). Top and bottom panels are the same image. [Figure 4] FIG. 1: Testing of recombinant AAT for anti-SARS-CoV-2 activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0282] Detailed description of the drawings: Figure 1: Vector maps for the expression of recombinant proteins in Pichia pastoris. The first vector shown is pGAPZα, which contains the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The second vector is pPICZα, which contains the inducible alcohol oxidase I (AOX1) promoter.

[0283] Figure 2: Purification of supernatants containing recombinant AAT produced in Pichia pastoris by anion exchange chromatography (AEX). Prolastin, a human plasma-derived α1AT preparation, was also applied to SEC. 700 ml of P. pastoris supernatant from both the unmodified WT strain and the strain containing the AAT expression vector were applied to the column. Fractions were taken at 30 s intervals.

[0284] Figure 3: Western blot analysis of recombinant AAT fractions obtained from anion exchange chromatography (AEX). The top and bottom panels are the same image, the bottom panel has been cropped to avoid the strong signal of prolastin. The contrast of the image in the bottom panel has been increased. 10 µg of protein per lane was loaded for each fraction obtained from AEX, and 2.5 µg of prolastin protein was loaded in the control wells.

[0285] Figure 4: Testing of recombinant AAT for anti-SARS-CoV-2 activity. (A) Experiments were performed with recombinant AAT fractions 52 and 53 (curves with circles and squares), human serum-derived AAT prolastin (curve with diamonds) and DMSO (curve with triangles), respectively. Caco2 (colon cancer cells susceptible to SARS-CoV-2) were seeded, the medium was removed on day 1 and serum-free medium was added to the cells. Recombinant α1AT samples were solubilized in 10% DMSO in H2O and all compounds and controls were titrated. Compounds were added to the cells and incubated for 1 h at 37 °C. Cells were transduced with lentiviral SARS-CoV-2 pseudoparticles. After 48 h, the luciferase signal in cell lysates was measured. The readout provided represents pseudoparticle cell entry (y-axis) against increasing concentrations (log; x-axis) of protein administered to the cells. (B) Data from the experiment presented in (A) are shown plotting pseudoparticle cell invasion (y-axis) against increasing concentrations of protein administered to the cells (linear; x-axis). Fraction 52 (triangles) and fraction 53 (circles) are shown relative to prolastin (diamond shapes). EXAMPLES

[0286] The following examples are presented to illustrate certain, and potentially preferred, embodiments of the present invention and demonstrate practical applicability without limiting the scope.

[0287] Example 1: Exemplary scheme for preparation of recombinant AAT: The following general overview regarding the expression and preparation of recombinant AAT is provided.

[0288] 1. Fermentation Fermentation of yeast cultures modified to recombinantly express human AAT is typically carried out in a bioreactor. The yeast culture is stimulated to produce alpha-1-antitrypsin under optimal conditions in terms of temperature, oxygen concentration, substrate concentration, etc. In an embodiment, the process is terminated at the point where the synthesis rate of the yeast culture decreases. In an embodiment, this lasts for about 36 hours to 144 hours, preferably 48 hours to 120 hours, more preferably 72 hours to 96 hours.

[0289] 2. Purification The contents of the reactor can then be harvested, preferably a culture supernatant is obtained and subsequently processed. The harvested fermentate consists of water, yeast culture, residues of various substrates and auxiliary substances that stabilize the process. Pure alpha-1-antitrypsin must be separated from this mixture. This purification is carried out in various steps, including separation of the yeast culture supernatant and subsequent separation by chromatography columns. At the end of this process, alpha-1-antitrypsin is available in pure form.

[0290] 3. Save AAT, like all other proteins, can be subject to biological degradation processes without further treatment. This can be stopped by cooling at -80°C. However, this procedure is not optimal under some conditions. For this reason, the material can be optionally freeze-dried (lyophilized), which allows it to be stored and transported without loss of stability. The active material is returned to its liquid state, for example by adding water, immediately before each use. This process is state of the art and is used for almost all protein preparations.

[0291] 4. Packaging "Fill and finish" involves dividing into the desired individual doses (e.g., 100 mg) and sterile packaging. Depending on the final product, various packaging formats can be considered.

[0292] Examples 2 to 5: Examples 2 to 5 show a detailed workflow for the production of human recombinant AAT in Pichia pastoris. The workflow consists of transformation of Pichia pastoris host cell line with AAT expression plasmid, microscale cultivation, microscale recultivation, and selection of lead clones. A Pichia pastoris expression strain for recombinant production of human alpha-antitrypsin (rhAAT) was developed.

[0293] Example 2: Design and receipt of synthetic AAT gene and vector construction: The gene sequence encoding AAT was designed for expression in P. pastoris.

[0294] The gene encoding the AAT protein is according to SEQ ID NO: 4 (nucleotide sequence of the inserted target gene, without the flanking regions used for cloning purposes, with a stop codon):

[0295] The protein sequence of AAT is according to SEQ ID NO:5: EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERP FEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMINEQINTKSPLFMGKVVNPTQK

[0296] Upon receiving the optimized synthetic gene sequence, the target gene was cloned into an appropriate expression plasmid (here pPICZα was employed) via restriction enzyme sites, e.g., XhoI / XbaI.

[0297] After receiving the synthetic gene in the supplier's plasmid, the dried plasmid was solubilized and transformed into Escherichia coli (E. coli) TOP10F' cells (NEB-5 alpha competent E. coli, C2987I, NEB). The E. coli clone harbors the supplier's plasmid with the synthetic gene. The transformants were restreaked on agar plates and the plasmid was isolated by standard plasmid preparation procedures.

[0298] The synthetic gene from the backbone of the supplier's plasmid was obtained by digesting the plasmid with double restriction enzymes. The digested plasmid was loaded onto an agarose gel and separated by gel electrophoresis. The synthetic gene band was excised from the agarose gel, purified and eluted, ready for ligation.

[0299] The double digested synthetic gene is ligated into the double digested yeast expression plasmid. The transformants were restreaked on agar plates and the plasmids were isolated by standard plasmid preparation procedures. The synthetic gene was separated from the backbone of the yeast expression plasmid by double digestion with restriction enzymes. The band sizes of the digested plasmids were confirmed by agarose gel electrophoresis. Selected plasmids were also sequenced using appropriate primers.

[0300] Positive clones were then restreaked onto agar plates for larger plasmid preparations (for transformation into Pichia). The prepared plasmids were then desalted with a membrane (MCE membrane, 0.025 μM, Millipore VSWP01300) and the DNA concentration was determined spectrophotometrically and adjusted to approximately 1 μg / μL.

[0301] Example 3: Workflow in Pichia pastoris: All media components used in the experiments with P. pastoris during the growth phase, transformation phase, regeneration phase (during transformation), and storage of yeast colonies were certified animal-free with respect to direct content, direct contact, or potential contamination. The following media were employed: YPhyD liquid medium (1% yeast extract, 2% phytonuclease, 2% dextrose), YPhyD solid medium (as above plus 2% w / v agar, supplemented with antibiotics as needed), BMD liquid (1.34% YNB, 2% dextrose, 0.2 M sodium phosphate buffer pH 6.0, 4 x 10-5% biotin).

[0302] Electroporations for all transformations were performed using the Pichia pastoris X33 competent expression cell line, applying standard procedures and standard equipment for electroporation.

[0303] Example 4: Microscale culture in 96 deep-well plates For screening, single colonies were picked from the transformation plates and placed into single wells of 96-deep-well plates filled with optimized culture medium, and the corner wells of some plates were inoculated with mock strains to serve as the matrix for the analysis.

[0304] After an initial growth phase to generate biomass (BMD solids), expression from the AOX1 promoter was induced by adding an optimized liquid mixture containing a defined concentration of methanol. At defined time points, further induction with methanol was performed.

[0305] A total of 72 hours after the initial methanol introduction, all deep-well plates were centrifuged and the supernatants of all wells were collected into stock microtiter plates for subsequent analysis. After selecting specific strains from the screening results, these strains were restreaked onto non-selective agar plates to yield single isolated colonies per strain. For each strain cultivated in the rescreen, six of these individual colonies were inoculated into a single well of a 96-deep-well plate filled with optimized culture medium and processed as described above.

[0306] Example 5: Analysis of target protein expression Supernatants were assessed for target protein expression based on molecular weight comparison of samples run on SDS-PAGE gels and Western blots. A previously prepared AAT sample was used as a control.

[0307] For SDS-PAGE, diluted or undiluted samples were mixed with 4x LDS sample buffer and 10x Novex reducing agent (both from Thermo Scientific) and incubated at 70°C for 10 min. Samples were then loaded onto Bolt Bis-Tris 4%-12% gels and run with MES buffer. SeeBlue Plus2 Pre-Stained Standards were included as molecular weight markers (both from Thermo Scientific).

[0308] For Western blots, protein transfer from pre-run gels was performed by dry blotting using an iBlot 2 Gel TransferDevice with a dedicated Novex PVDF transfer stack, after which the membrane was saturated with PBS, 0.1% Tween-20, 5% BSA for 30 min at room temperature with gentle shaking.

[0309] For AAT specific detection, the membrane was incubated with the primary AAT antibody (SIGMA, SAB4200196-200 μL) diluted 1:750 in PBS, 0.1% Tween-20, 3% BSA for 30 min at room temperature with gentle shaking. After three washing steps (5 s, 5 min and 10 min, PBS-0.1% Tween20, with vigorous agitation at approximately 200 rpm), the secondary anti-mouse antibody (SIGMA Anti-Mouse IgG-Peroxidase A2554-1 mL) diluted 1:2000 in PBS, 0.1% Tween-20, 1% BSA was added for 20 min. The membrane was then washed again and developed by adding TMB ultra substrate (Thermo Scientific).

[0310] SDS-PAGE gel and Western blot analysis revealed that AAT was expressed at a molecular weight similar to that of standard preparations.

[0311] Additionally, the supernatants were assessed for target protein expression based on microfluidic capillary electrophoresis separation.

[0312] A method was established that involves microfluidic capillary electrophoretic separation (GXII, CaliperLS, now Perkin Elmer) followed by identification of the target protein based on its size. Briefly, a few μL of all culture supernatants are fluorescently labeled and analyzed according to protein size using a microfluidics-based electrophoresis system. An internal standard sample (contained in a solution provided by the supplier) allows an approximate assignment to the size in kDa and the approximate concentration of the detected signal. Bovine serum albumin (BSA) was used as a calibrator of apparent molecular weight and concentration after dilution to known concentrations in mock strain matrix.

[0313] Procedure: 5 μL of sample (from deglycosylation, see below) was mixed with 8 μL of sample buffer (Perkin Elmer, with LDS, pH 7.58) containing an appropriate amount of reducing agent (pH 7.37 now) and heated for 5 min at 95° C. Subsequently, 32 μL of ddH2O was added and after centrifugation at 4000 rpm for 3 min the sample was applied to the mCE (to pellet potential aggregates).

[0314] The estimated concentration of rhAAT in the supernatant was calculated by comparing the specific peak area with that of BSA, which was present at known concentrations. Various yeast clones were tested and showed estimated rhAAT amounts ranging from 2 mg / mL to 11 mg / mL.

[0315] Example 6: Testing the antiprotease activity of recombinant AAT produced in Pichia pastoris Compound: Prolastin: AAT derived from human serum Camostat mesylate: A trypsin-like serine protease inhibitor. rhAAT: recombinant AAT produced in Pichia pastoris

[0316] Materials and Methods The respective protease (TMPRSS2 or neutrophil elastase) is mixed with serial dilutions of rhAAT, camostat mesilate or prolastin, which results in the formation of a complex between the test compound and the protease. A fluorescent reporter peptide substrate is then added. Upon cleavage of the reporter peptide substrate by the protease, a fluorescent dye is released. Thus, the fluorescence intensity serves as a signal of the remaining protease activity.

[0317] TMPRSS2 activity assay: To assess the activity of recombinant human TMPRSS2, 25 μL of serially diluted prolastin, camostat mesylate, or rhAAT are incubated with 25 μl of 2 μg / ml recombinant TMPRSS2 enzyme (LSBio #LS-G57269) in assay buffer (50 mM Tris-HCL, 0.154 mM NaCl pH 8.0) for 15 min at 37° C. Then, 50 μl of 20 μM BOC-Gln-Ala-Arg-AMC protease substrate (Bachem #4017019) is added and incubated for 2 h at 37° C. Fluorescence intensity is measured after 2 h at an excitation wavelength of 380 nm and an emission wavelength of 460 nm in a Synergy™ H1 microplate reader (BioTek) using Gen5 3.04 software. The assay is performed in a flat-bottom 96-well plate.

[0318] Neutrophil elastase activity assay: Neutrophil elastase activity is measured by mixing 25 μL of serially diluted prolastin, camostat mesylate, or rhAAT with 25 μL of 2 ng / μl recombinant neutrophil elastase (Merck Millipore #324681) in assay buffer (50 mM Tris, 1 M NaCl, 0.05% (w / v) Brij-35, pH 7.5) for 15 min at 37° C. Then, 50 μl of 200 μM MEOSUC-Ala-Ala-Pro-Val-AMC substrate (Bachem #4005227) is added and incubated at 37° C. Fluorescence intensity was measured after 5 min at an excitation wavelength of 380 nm and an emission wavelength of 460 nm in a Synergy™ H1 microplate reader (BioTek) using Gen5 3.04 software. Assays are performed in flat-bottom 96-well plates.

[0319] result: All three test compounds inhibit the activity of TMPRSS2 and neutrophil elastase. IC50 analysis of rhAAT, prolastin, and camostat mesylate is performed. rhAAT inhibits TMPRSS2 proteolytic activity in a dose-dependent manner with an IC50 of approximately 350 nM.

[0320] Example 7: Testing anti-SARS-CoV-2 activity by pseudovirus assay Introduction: Infection of target cells by viruses is governed by viral surface glycoproteins. Viral pseudoparticles are engineered viruses that carry foreign viral glycoproteins (gp) on their surface. For example, the pseudoparticles used in this study are based on HIV-1 virions covered with the spike gp of SARS-CoV-2 or the gp of vesicular stomatitis virus (VSV-G). Thus, they enter cells by the same mechanism as SARS-CoV-2 or VSV-G, respectively. However, pseudoviruses lack genetic information important for replication and instead deliver reporter genes to infected cells. Thus, pseudoparticles allow for the safe and high-throughput investigation of highly pathogenic viruses.

[0321] Materials and Methods: To test the activity of rhAAT against SARS-CoV-2 pseudoparticles, cells are treated with serial dilutions of rhAAT, prolastin, and camostat mesylate to allow complex formation between rhAAT and TMPRSS2. Cells are then inoculated with pseudoparticles carrying SARS-CoV-2 spike gp or VSV-G, and viral entry is quantified after 2 days by measuring reporter gene activity.

[0322] Generation of lentiviral pseudoparticles: For the generation of lentiviral SARS-CoV-2 (LV(Luc)-CoV-2) pseudoparticles, 900,000 HEK293T cells are seeded in 6-well plates in DMEM medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin. The next day, cells were transfected with 0.49 μg of pCMVdR8_91 (encoding a replication-deficient lentivirus), 0.49 μg of pSEW-Luc2 (encoding a luciferase reporter gene, both kindly provided by Prof. Christian Buchholz, Paul Ehrlich Institute, Germany), and 0.02 μg of either pCG1-SARS-2-SΔ18(WT), pcDNA3_1 SARS-CoV-2-Sd19B.1.617.2_4377 (B.1.617.2, Delta) or pCG1_SARS-2-SΔ18 (BA.4 and BA.5, Omicron) by mixing the plasmid DNA with PEI in a 1:3 ratio in serum-free medium.

[0323] After 20 min incubation at room temperature, the transfection mix is ​​added dropwise to the cells. 8 h post-transfection, the cells are washed and growth medium containing 2.5% FCS is added. 48 h post-transfection, the pseudoparticle containing supernatant is harvested and clarified by centrifugation at 450 g for 5 min. The virus stock is aliquoted and stored at -80 °C until use.

[0324] Pseudoparticle Inhibition Assay: One day before transduction, 10,000 Caco2 cells were seeded in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U / l penicillin, 100 mg / ml streptomycin, 1× non-essential amino acids, and 1 mM sodium pyruvate in a 96-well flat-bottom plate. The next day, the medium was replaced with 60 μl of serum-free growth medium, and the cells were treated with serially diluted rhAAT, prolastin, and camostat mesilate at 37° C. for 1 h, followed by transduction of the cells with 20 μl of each lentiviral pseudoparticle. VSV-G spike lentiviral pseudoparticles served as a negative control.

[0325] The transduction rate is assessed by measuring the luciferase activity in cell lysates 48 hours after transduction using a commercial kit (Luciferase Assay System, Promega) in an Orion II microplate reader with simplicity 4.2 software. The value of the untreated control is set to 100% transduction.

[0326] result: rhAAT inhibits SARS-CoV-2 entry as well as prolastin and camostat mesylate. VSV-G pseudoparticles were not affected by the test compounds and were able to enter the cells. rhAAT is non-toxic to Caco-2 cells at the tested concentrations. This experiment shows a comparative analysis of pseudoparticle entry in Caco2 cells treated with rhAAT, prolastin and camostat mesylate, respectively. Cells treated with rhAAT at a concentration of 20 mg / ml show almost no pseudoparticle entry.

[0327] Examples 6 and 7 are based in part on the experimental setup described in Azouz et al. 2021 (Pathog Immun. 2021 Apr 26; 6(1):55-74).

[0328] Example 8: In vitro treatment of human airway epithelial cells infected with SARS-CoV2 with prolastin and purified recombinant AAT produced in Pichia pastoris This example is designed to test whether recombinant AAT produced in Pichia pastoris inhibits SARS-CoV2 replication in human primary airway epithelial cells more effectively than prolastin. The experimental setup is based on Wettstein et al. 2021 (Nat Commun 12, 1726).

[0329] Small airway epithelial cells (SAECs) were infected with SARS-CoV-2 and then treated with prolastin and recombinant AAT separately. The inoculum was immediately removed, and supernatants were collected several days after infection and subjected to SARS-CoV-2-specific RT-qPCR.

[0330] Human airway epithelial cells (HAECs) were exposed to prolastin and recombinant AAT separately and then inoculated with SARS-CoV-2. Cells were fixed on days 1, 2, and 3 post-infection and stained with DAPI, SARS-CoV-2-specific spike antibody, and α-tubulin-specific antibody.

[0331] Human small airway epithelial cells: Human small airway epithelial cells (Lonza, CC-2547, batch: 18TL082942, donor: 68 years old, female) are cultured in SAGM™ small airway epithelial cell growth medium (Lonza, CC-3118). Alternatively, differentiated air-liquid interface cultures of human airway epithelial cells (HAECs) are generated from primary human basal cells isolated from airway epithelium.

[0332] Cells are expanded in T75 flasks (Sarstedt) in Airway Epithelial Cell Basal Medium supplemented with Airway Epithelial Cell Growth Medium SupplementPack (both from Promocell). Growth medium is changed every 2 days. Upon reaching 90% confluence, HAECs are detached using a DetachKIT (Promocell) and seeded onto 6.5 mm Transwell filters (Corning Costar). Filters are pre-coated overnight with collagen solution (StemCell Technologies) and exposed to UV light for 30 min prior to cell seeding to cross-link and sterilize the collagen. 3.5 × 10 cells in 200 μl of growth medium are added. 4 10 cells are added to the apical side of each filter, and an additional 600 μl of growth medium is added to the basolateral side. The apical medium is replaced after 48 h. After 72–96 h, when the cells reach confluence, the apical medium is removed and the basolateral medium is switched to differentiation medium. Differentiation medium consists of a 1:1 mixture of DMEM-H supplemented with Airway Epithelial Cell Growth Medium SupplementPack and LHC Basal (ThermoFisher), which is replaced every 2 days. Air lifting (removal of apical medium) was defined as day 0 of air-liquid interface (ALI) culture, and cells were grown in ALI conditions until experiments were performed on days 25–28. To avoid accumulation of mucus on the apical side, HAEC cultures are washed apically with PBS for 30 min every 3 days from day 14 onward.

[0333] SARS-CoV-2 infection of HAEC: Immediately prior to infection, the apical surface of HAEC grown on Transwell filters is washed three times with 200 μl of PBS to remove accumulated mucus. 10 μM α1AT or 5 μM remdesivir is then added to the basal medium and apical surface. Cells are then incubated with 9.25 × 10 2The cells were infected with 100 plaque-forming units (PFU) of SARS-CoV-2 (BetaCoV / France / IDF0372 / 2020). After 2 h of incubation at 37 °C, the viral inoculum was removed and the cells were washed three times with 200 μl of PBS and cultured at the air-liquid interface again. On days 1, 2 and 3 post-infection, the cells were fixed with 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.2% saponin and 10% FCS in PBS for 10 min, washed twice with PBS and stained overnight at 4 °C with anti-SARS-CoV-2 spike (ab252690, Abcam) and anti-α-tubulin (MA1-8007, Thermo Scientific) diluted 1:300–1:500 in PBS, 0.2% saponin and 10% FCS, respectively.

[0334] Subsequently, cells are washed twice with PBS and incubated for 1 h at room temperature in PBS, 0.2% saponin and 10% FCS containing AlexaFluor 488-labeled anti-rabbit and AlexaFluor 647-labeled anti-rat secondary antibodies (all 1:500; Thermo Scientific) and DAPI + phalloidin AF 405 (1:5000; Thermo Scientific). Images are taken on an inverted confocal microscope (Leica TCS SP5, Leica Microsystems, Leica application suite version 2.7.3.9723) using a 40x lens (LeicaHC PL APO CS2 40x1.25 OIL). Images of blue (DAPI), green (AlexaFluor 488) and far-red (AlexaFluor 647) channels are taken in sequential mode using appropriate excitation and emission settings that were kept constant for all acquisitions. For quantification, randomly chosen locations in each filter were selected and z-stacks were acquired. A maximum z-projection was performed, and the area per unit area (0.15 mm 2 ) anti-SARS-CoV-2 positive cells were visually identified and counted.

[0335] result: Recombinant AAT inhibits SARS-CoV-2 replication in primary human airway cells more effectively than prolastin. Recombinant AAT present during infection reduces viral titers more than prolastin. SARS-CoV-2 spike expression is less detectable in infected recombinant AAT-treated HAECs than in prolastin-treated HAECs.

[0336] Example 9: Cure study using AAT inhalation: In the context of the individual self-study, patients were informed of the protocol and then treated by inhalation with AAT (Prolastin, Grifols, Spain). A Pari Boy Pro (Pari, Starnberg, Germany) was used as the nebulizer, whereby 100 mg of AAT was dissolved in 8 ml of saline and added to the nebulizer container.

[0337] Patient 1, male, 52 years old, with smoking risk factors, unvaccinated During the course of treatment, On day 1, patient 1 tested positive for Covid-19 by PCT and had mild symptoms similar to influenza-like infection. On day 2, patient 1 tested negative for Covid-19 by rapid test and was asymptomatic. The patient inhaled 100 mg of Prolastin. Between days 3 and 5, patient 1 tested negative for Covid-19 by rapid test and was asymptomatic.

[0338] Patient 2, female, 25 years old, with smoking risk factor Patient 2 had received two doses of the Comirnaty vaccine, the second of which was administered three months before contracting Covid-19. On day 1, patient 2 tested positive for Covid-19 twice by rapid test and had fever, sore throat, fatigue, and decreased taste. Patient 2 inhaled 100 mg of AAT three times, 4 hours apart. Symptoms were significantly improved on day 2. Patient 2 inhaled 100 mg of AAT on day 2. On day 3, patient 2 was asymptomatic and inhaled 100 mg of AAT. Patient 2 tested negative for Covid-19 by PCR.

[0339] Example 10: Recombinant AAT Production from Pichia pastoris: As an alternative to Examples 2 to 5, the following experimental validation was performed on recombinant AAT produced in Pichia pastoris.

[0340] A codon-optimized AAT gene for expression in P. pastoris was designed using GENEius from Eurofins provided in the pEX-A258 vector.

[0341] The codon-optimized gene encoding the AAT protein is according to SEQ ID NO:2.

[0342] The protein sequence of AAT is according to SEQ ID NO:3. EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERP FEVKDATEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMINEQNTKSPLFMGKVVNPTQK

[0343] After restriction enzyme digestion and subsequent cloning, the AAT gene was inserted into an expression vector for expression in Pichia pastoris. Two suitable expression vectors are shown in Figure 1. The first vector shown is pGAPZα, which contains the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The second vector is pPICZα, which contains the inducible alcohol oxidase I (AOX1) promoter.

[0344] The constructs were cloned into the respective vectors pGAPzα and pPICZα using Gibson assembly, followed by directed mutagenesis using the Phusion Site-Directed Mutagenesis Kit (ThermoFischer) to delete the 33 bp linker and His tag and produce untagged AAT, generating both the His-tagged AAT fusion protein and the untagged AAT construct.

[0345] Pichia pastoris X33 was then transformed with the AAT expression vector and cultured under standard conditions.

[0346] Fermentation is carried out in a bioreactor using mineral salts medium FM22 (Stratton et al1998, high cell-density fermentation, Methods Mol Biol.), which may contain various salts and 40 g / L glycerol as the main carbon source. The environmental parameters defined in the bioreactor are: 30°C, pH 6, 1 vvm gas, dissolved oxygen concentration 30%. All parameters are adjusted accordingly in the bioreactor.

[0347] To increase the cell concentration, glycerol feeding is started as soon as the first batch growth is completed (after approximately 24 hours). In that case, the increase in cell concentration leads to an increase in AAT production. When pGAPz is used, AAT is already produced during growth (constitutive) and only needs to be harvested (after 3 days). When pPICz is used, the glycerol feeding is followed by another methanol feeding to induce the promoter and start AAT synthesis. This process takes a correspondingly long time (4 days). In one embodiment, the fermentation with pGAPz and glycerol feeding had a cell concentration of 42 g / L (dry weight), a total protein content of 115 mg / L and an AAT concentration of 15 mg / L.

[0348] After the fermentation process is complete, a supernatant is obtained by first centrifuging the cells. Optionally, the clear supernatant is concentrated by ultrafiltration (10 kDa or 30 kDa cutoff), leaving the AAT and other proteins in the retentate. The filtrate can be discarded. The retentate can be purified using any suitable method, for example, metal affinity chromatography, His-tag purification (if a His-tag construct is employed), or anion exchange chromatography.

[0349] Example 11: Purification of AAT from the supernatant of transformed Pichia pastoris X33: Pichia pastoris X33 was engineered to recombinantly express α1AT in the culture supernatant (SN) as described above. SN of P. pastoris expressing α1AT (recombinant α1AT) and unmodified P. pastoris SN were purified by anion exchange chromatography (AEX).

[0350] To gain experience with the elution profile of α1AT and to see whether SEC affects the activity of α1AT, Prolastin, a human plasma-derived α1AT preparation, was also applied to SEC. 700 ml of P. pastoris supernatant was applied to the column and fractions were obtained at 30 s intervals, resulting in two fractions per minute.

[0351] result: The elution peaks of SN from prolastin and recombinant α1AT are different, but the reason for the difference is unclear. Differences in the glycosylation patterns of human and yeast AAT may be responsible for the different elution profiles. WT P. pastoris SN results in a broad peak in AEX. SN from recombinant α1AT shows additional peaks not present in WT SN, suggesting the presence of recombinant protein expression. The elution profile is shown in Figure 2. The additional peaks (~fraction 52 and fraction 53) were assumed to contain recombinant α1AT and were used for further assays.

[0352] Example 12: Western blot analysis of recombinant AAT fractions Recombinant AAT was analyzed by Western blot. BCA assay was performed on samples. Samples were boiled at 70°C for 10 min and run on a 4%-12% SDS gel at 120 V for 90 min. Samples were blotted onto a PVDF membrane (semi-dry) and stained with primary antibody (16382-1-AP, rabbit, 1:1000 dilution) overnight at 4°C. After washing the membrane, secondary antibody was added and incubated at room temperature for 30 min. After washing the membrane was imaged.

[0353] result: As shown in Figure 3, recombinant AAT is visible via Western blot analysis in fractions 52 to 57. Of note, rhAAT provides a well-defined band, whereas prolastin exhibits a smear due to multiple by-products / impurities, suggesting that rhAAT production represents an improvement in the preparation of AAT.

[0354] Example 13: Testing of recombinant AAT for anti-SARS-CoV-2 activity Compound solubilization: Prolastin (not purified by CEX) was solubilized in H O. Fractions presumed to contain recombinant α1AT were solubilized in 10% DMSO.

[0355] Transduction protocol: On day 0, 10,000 Caco2 (colon cancer cells susceptible to SARS-CoV-2) were seeded. On day 1, the medium was removed and serum-free medium was added to the cells. Recombinant α1AT samples were solubilized in 10% DMSO in H2O and all compounds and controls were titrated. Compounds were added to the cells and incubated for 1 h at 37°C. Cells were transduced with lentiviral SARS-CoV-2 pseudoparticles. After 48 h, luciferase signals were measured in cell lysates.

[0356] The tests used lentiviral pseudoparticles carrying the SARS-CoV-2 spike protein of the Wuhan Hu-1 isolate. Pseudoparticles are replication-defective viral particles that display the glycoprotein of another virus. They are used to study viral entry and often carry a reporter gene (here luciferase) that is expressed when the virus enters a cell.

[0357] Table. IC50 values ​​for inhibition of pseudoparticle entry into Caco2 cells after administration of AAT or control: [Table 2]

[0358] result: Recombinant α1AT fractions 52 and 53 inhibit SARS-CoV-2 entry similarly to prolastin, suggesting that recombinant α1AT is present and active in these fractions. As shown in Figure 4, a comparative analysis shows pseudoparticle entry in Caco2 cells treated with recombinant AAT fractions 52 and 53 (curves with circles and squares), human serum-derived AAT prolastin (curves with diamonds) and DMSO (curves with triangles), respectively. At a concentration of 20 mg / ml, cells treated with recombinant AAT completely inhibit pseudoparticle entry, while approximately 10% pseudoparticle entry occurs in cells treated with prolastin. Figures 4A and 4B show greater inhibition of pseudoparticle entry by fractions 52 and 53 compared to prolastin at protein concentrations of 10 mg / mL and 20 mg / mL.

[0359] It is noteworthy that the inhibition curves of prolastin and rhAAT have different shapes. For prolastin, a clearly more nonspecific inhibition is observed as can be seen from the curve shape, where nonspecific inhibition begins at low concentrations and does not reach complete inhibition at higher doses. For rhAAT, a steeper curve shape with a clear inflection point is observed, suggesting a more specific inhibition by rhAAT. Prolastin may be less specific or contain additional impurities, leading to a nonspecific inhibitory effect.

[0360] As can be derived from the above preliminary IC50 values, fraction 53 with AAT isolated from P. pastoris shows greater activity in the employed pseudoparticle assay compared to prolastin. Tests using AAT from different P. pastoris expression systems, using controlled AAT concentrations and different purification techniques, compared to prolastin, are ongoing.

[0361] Additional evaluation of rhAAT compared to Prolastin is underway and IC90 values ​​(also highly relevant to viral inhibition assays) may play a role in assessing rhAAT activity. From initial assessments, IC90 values ​​appear to differ for Prolastin and rhAAT as derived from Figure 4. Initial findings derived from this assay reveal an estimated IC90 of 20 mg / mL for Prolastin and 8 mg / mL for rhAAT, again suggesting an improvement of rhAAT over Prolastin.

[0362] Drawing translation Figure 1 α-factor myc epitope myc epitope Stop Stop Zeocin c-mycepitope c-myc epitope Figure 2 rec.α1AT supernatant Recombinant α1AT supernatant mock supernatant Prolastin Figure 3 Regular image Prolastin increased contrast(cut prolastin (high signal)) Figure 4 A Pseudoparticle entry (%) Compound (mg / ml) Compound (mg / ml) rec.α1AT Fraction 53 Recombinant α1AT Fraction 53 rec.α1AT Fraction 52 Recombinant α1AT Fraction 52 Prolastin DMSO ctrl DMSO control B Pseudoparticle entry (%) Concentration(mg / ml) Recombinant AAT sample 52 Recombinant AAT sample 53 Prolastin

Claims

1. A medicament for treating viral respiratory infections, comprising a recombinant alpha 1-antithricin (AAT) protein or a fragment thereof expressed in a genetically modified yeast.

2. The pharmaceutical composition of claim 1, wherein the viral respiratory infection is SARS coronavirus infection.

3. The pharmaceutical of claim 2, wherein the viral respiratory infection is SARS-CoV-2.

4. The pharmaceutical composition of claim 1, wherein the viral respiratory infection is influenza or respiratory syncytial virus (RSV).

5. The method of claim 1, wherein the AAT protein or a fragment thereof comprises a sequence according to SEQ ID NO: 3 or SEQ ID NO: 5, or is encoded by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

4.

6. The pharmaceutical composition according to claim 1, wherein the recombinant AAT or a fragment thereof has a serum half-life and / or activity equal to or greater than that of AAT purified from human plasma.

7. The pharmaceutical of claim 1, wherein the recombinant AAT protein or a fragment thereof is expressed in a yeast of the Saccharomycete family.

8. The method of claim 1, wherein the recombinant AAT protein or a fragment thereof is expressed in Pichia pastoris.

9. The pharmaceutical composition of claim 1, comprising a post-translational modification of the recombinant AAT protein or a fragment thereof.

10. The pharmaceutical according to claim 9, wherein the post-translational modification is O-glycosylation, N-glycosylation, N-terminal methionine removal, N-acetylation and / or phosphorylation, or any combination thereof.

11. The method of claim 1, wherein the AAT protein or a fragment thereof is administered by inhalation.

12. The pharmaceutical composition of claim 11, wherein the AAT protein or fragment thereof is administered by inhalation as a solution suitable for inhalation, a soluble powder suitable for inhalation, or a dry powder suitable for inhalation.

13. The method of claim 12, wherein the AAT protein or fragment thereof is administered by inhalation of a nebulized solution.

14. 2. The method of claim 1, wherein the viral infection is SARS-CoV-2 and the AAT protein or a fragment thereof is administered by inhalation of a nebulized solution.

15. 2. The pharmaceutical composition of claim 1, wherein the viral infection is SARS-CoV-2, influenza, or RSV, the AAT protein or a fragment thereof is expressed from Pichia pastoris, and the AAT protein or a fragment thereof is administered by inhalation of a nebulized solution.

16. 2. The pharmaceutical composition of claim 1, wherein the viral infection is SARS-CoV-2, the AAT protein or a fragment thereof is expressed from Pichia pastoris and isolated using chromatography, such as ion exchange chromatography or hydrophobic interaction chromatography, and the AAT protein or a fragment thereof is administered by inhalation of a nebulized solution.

17. The AAT protein or a fragment thereof a. culturing a genetically modified yeast comprising an exogenous nucleic acid molecule having an AAT coding region operably linked to a promoter or promoter / enhancer combination; b. expressing the recombinant AAT or a fragment thereof in the cultured yeast; c. isolating the recombinant AAT or a fragment thereof from the culture; The pharmaceutical of claim 1, which is produced from a genetically modified yeast in a method comprising:

18. 18. The pharmaceutical of claim 17, wherein the AAT protein or fragment thereof is secreted from the yeast into the culture medium at a concentration of at least 1 mg of protein per liter of liquid medium (mg / L).

19. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a recombinant AAT protein or a fragment thereof and a pharmaceutically acceptable carrier.

20. 20. The medicament of claim 19, wherein the composition is a solution suitable for inhalation, a soluble powder suitable for inhalation, or a dry powder suitable for inhalation.

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